Beta 1,3-galactosyltransferases from C. jejuni

ABSTRACT

This invention provides prokaryotic glycosyltransferases, including a bifunctional sialyltransferase that has both an α2,3- and an α2,8-activity. A β1,4-GalNAc transferase and a β1,3-galactosyltransferase are also provided by the invention, as are other glycosyltransferases and enzymes involved in synthesis of lipooligosaccharide (LOS). The glycosyltransferases can be obtained from, for example,  Campylobacter  species, including  C. jejuni.  In additional embodiments, the invention provides nucleic acids that encode the glycosyltransferases, as well as expression vectors and host cells for expressing the glycosyltransferases.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional ApplicationNo. 60/118,213, which was filed on Feb. 1, 1999, and is acontinuation-in-part of U.S. application Ser. No. 09/495,406 filed Jan.31, 2000, both of which are incorporated herein by reference for allpurposes.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention pertains to the field of enzymatic synthesis ofoligosaccharides, including gangliosides and ganglioside mimics.

[0004] 2. Background

[0005] Gangliosides are a class of glycolipids, often found in cellmembranes, that consist of three elements. One or more sialic acidresidues are attached to an oligosaccharide or carbohydrate core moiety,which in turn is attached to a hydrophobic lipid (ceramide) structurewhich generally is embedded in the cell membrane. The ceramide moietyincludes a long chain base (LCB) portion and a fatty acid (FA) portion.Gangliosides, as well as other glycolipids and their structures ingeneral, are discussed in, for example, Lehninger, Biochemistry (WorthPublishers, 1981) pp. 287-295 and Devlin, Textbook of Biochemistry(Wiley-Liss, 1992). Gangliosides are classified according to the numberof monosaccharides in the carbohydrate moiety, as well as the number andlocation of sialic acid groups present in the carbohydrate moiety.Monosialogangliosides are given the designation “GM”,disialogangliosides are designated “GD”, trisialogangliosides “GT”, andtetrasialogangliosides are designated “GQ”. Gangliosides can beclassified further depending on the position or positions of the sialicacid residue or residues bound. Further classification is based on thenumber of saccharides present in the oligosaccharide core, with thesubscript “1” designating a ganglioside that has four saccharideresidues (Gal-GalNAc-Gal-Glc-Ceramide), disaccharide (Gal-Glc-Ceramide)and monosaccharide (Gal-Ceramide) gangliosides, respectively.

[0006] Gangliosides are most abundant in the brain, particularly innerve endings. They are believed to be present at receptor sites forneurotransmitters, including acetylcholine, and can also act as specificreceptors for other biological macromolecules, including interferon,hormones, viruses, bacterial toxins, and the like. Gangliosides are havebeen used for treatment of nervous system disorders, including cerebralischemic strokes. See, e.g., Mahadnik et al. (1988) Drug DevelopmentRes. 15: 337-360; U.S. Pat. Nos. 4,710,490 and 4,347,244; Horowitz(1988) Adv. Exp. Med. and Biol. 174: 593-600; Karpiatz et al. (1984) AdvExp. Med. and Biol. 174: 489-497. Certain gangliosides are found on thesurface of human hematopoietic cells (Hildebrand et al. (1972) Biochim.Biophys. Acta 260: 272-278; Macher et al. (1981) J. Biol. Chem. 256:1968-1974; Dacremont et al. Biochim. Biophys. Acta 424: 315-322; Klocket al. (1981) Blood Cells 7: 247) which may play a role in the terminalgranulocytic differentiation of these cells. Nojiri et al. (1988) J.Biol. Chem. 263: 7443-7446. These gangliosides, referred to as the“neolacto” series, have neutral core oligosaccharide structures havingthe formula [Galβ-(1,4)GlcNAcβ(1,3)]_(n)Galβ(1,4)Glc, where n=1-4.Included among these neolacto series gangliosides are 3′-nLM₁(NeuAcα(2,3)Galβ(1,4)GlcNAcβ(1,3)Galβ(1,4)-Glcβ(1,1)-Ceramide) and6′-nLM₁ (NeuAcα(2,6)Galβ(1,4)GlcNAcβ(1,3)Galβ(1,4)-Glcβ(1,1)-Ceramide).

[0007] Ganglioside “mimics” are associated with some pathogenicorganisms. For example, the core oligosaccharides oflow-molecular-weight LPS of Campylobacter jejuni O:19 strains were shownto exhibit molecular mimicry of gangliosides. Since the late 1970s,Campylobacter jejuni has been recognized as an important cause of acutegastroenteritis in humans (Skirrow (1977) Brit. Med. J. 2:9-11).Epidemiological studies have shown that Campylobacter infections aremore common in developed countries than Salmonella infections and theyare also an important cause of diarrheal diseases in developingcountries (Nachamkin et al. (1992) Campylobacter jejuni: Current Statusand Future Trends. American Society for Microbiology, Washington, D.C.).In addition to causing acute gastroenteritis, C. jejuni infection hasbeen implicated as a frequent antecedent to the development ofGuillain-Barré syndrome, a form of neuropathy that is the most commoncause of generalyzed paralysis (Ropper (1992) N. Engl. J. Med. 326:1130-1136). The most common C. jejuni serotype associated withGuillain-Barré syndrome is O:19 (Kuroki (1993) Ann. Neurol. 33: 243-247)and this prompted detailed study of the lipopolysaccharide (LPS)structure of strains belonging to this serotype (Aspinall et al. (1994a)Infect. Immun. 62: 2122-2125; Aspinall et al. (1994b) Biochemistry 33:241-249; and Aspinall et al. (1994c) Biochemistry 33: 250-255).

[0008] Terminal oligosaccharide moieties identical to those of GD1a,GD3, GM1 and GT1a gangliosides have been found in various C. jejuni O:19strains. C. jejuni OH4384 belongs to serotype O:19 and was isolated froma patient who developed the Guillain-Barré syndrome following a bout ofdiarrhea (Aspinall et al. (1994a), supra.). It was showed to possess anouter core LPS that mimics the tri-sialylated ganglioside GT1a.Molecular mimicry of host structures by the saccharide portion of LPS isconsidered to be a virulence factor of various mucosal pathogens whichwould use this strategy to evade the immune response (Moran et al.(1996a) FEMS Immunol. Med. Microbiol. 16: 105-115; Moran et al. (1996b)J. Endotoxin Res. 3: 521-531).

[0009] Consequently, the identification of the genes involved in LPSsynthesis and the study of their regulation is of considerable interestfor a better understanding of the pathogenesis mechanisms used by thesebacteria. Moreover, the use of gangliosides as therapeutic reagents, aswell as the study of ganglioside function, would be facilitated byconvenient and efficient methods of synthesizing desired gangliosidesand ganglioside mimics. A combined enzymatic and chemical approach tosynthesis of 3′-nLM, and 6′-nLM₁ has been described (Gaudino and Paulson(1994) J. Am. Chem. Soc. 116: 1149-1150). However, previously availableenzymatic methods for ganglioside synthesis suffer from difficulties inefficiently producing enzymes in sufficient quantities, at asufficiently low cost, for practical large-scale ganglioside synthesis.Thus, a need exists for new enzymes involved in ganglioside synthesisthat are amenable to large-scale production. A need also exists for moreefficient methods for synthesizing gangliosides. The present inventionfulfills these and other needs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIGS. 1A-1C show lipooligosaccharide (LOS) outer core structuresfrom C. jejuni O:19 strains. These structures were described by Aspinallet al. (1994) Biochemistry 33, 241-249, and the portions showingsimilarity with the oligosaccharide portion of gangliosides aredelimited by boxes. FIG. 1A: LOS of C. jejuni O:19 serostrain (ATCC#43446) has structural similarity to the oligosaccharide portion ofganglioside GD1a. FIG. 1B: LOS of C. jejuni O:19 strain OH4384 hasstructural similarity to the oligosaccharide portion of gangliosideGT1a. FIG. 1C: LOS of C. jejuni OH4382 has structural similarity to theoligosaccharide portion of ganglioside GD3.

[0011]FIGS. 2A-2B show the genetic organization of the cst-I locus fromOH4384 and comparison of the LOS biosynthesis loci from OH4384 and NCTC11168. The distance between the scale marks is 1 kb. FIG. 2A shows aschematic representation of the OH4384 cst-I locus, based on thenucleotide sequence which is available from GenBank (#AF 130466). Thepartial prfB gene is somewhat similar to a peptide chain release factor(GenBank #AE000537) from Helicobacter pylori, while the cysD gene andthe partial cysN gene are similar to E. coli genes encoding sulfateadenylyltransferase subunits (GenBank #AE000358). FIG. 2B shows aschematic representation of the OH4384 LOS biosynthesis locus, which isbased on the nucleotide sequence from GenBank (#AF130984). Thenucleotide sequence of the OH4382 LOS biosynthesis locus is identical tothat of OH4384 except for the cgtA gene, which is missing an “A” (seetext and GenBank #AF167345). The sequence of the NCTC 11168 LOSbiosynthesis locus is available from the Sanger Centre(URL:http//www.sanger.ac.uk/Projects/C_jejuni/). Correspondinghomologous genes have the same number with a trailing “a” for the OH4384genes and a trailing “b” for the NCTC 11168 genes. A gene unique to theOH4384 strain is shown in black and genes unique to NCTC 11168 are shownin grey. The OH4384 ORF's #5a and #10a are found as an in-frame fusionORF (#5b/10b) in NCTC 11168 and are denoted with an asterisk (*).Proposed-functions for each ORF are found in Table 4.

[0012]FIG. 3 shows an alignment of the deduced amino acid sequences forthe sialyltransferases. The OH4384 cst-I gene (first 300 residues),OH4384 cst-II gene (identical to OH4382 cst-II), O:19 (serostrain)cst-II gene (GenBank #AF167344), NCTC 11168 cst-II gene and an H.influenzae putative ORF (GenBank #U32720) were aligned using theClustalX alignment program (Thompson et al. (1997) Nucleic Acids Res.25, 4876-82). The shading was produced by the program GeneDoc (Nicholas,K B., and Nicholas, H. B. (1997) URI:http://www.cris.com/˜ketchup/genedoc.shtml).

[0013]FIG. 4 shows a scheme for the enzymatic synthesis of gangliosidemimics using C. jejuni OH4384 glycosyltransferases. Starting from asynthetic acceptor molecule, a series of ganglioside mimics wassynthesized with recombinant α-2,3-sialyltransferase (Cst-I),β-1,4-N-acetylgalactosaminyltransferase (CgtA),β-1,3-galactosyltransferase (CgtB), and a bi-functionalα-2,3/α-2,8-sialyltransferase (Cst-II) using the sequences shown. Allthe products were analyzed by mass spectrometry and the observedmonoisotopic masses (shown in parentheses) were all within 0.02% of thetheoretical masses. The GM3, GD3, GM2 and GM1a mimics were also analyzedby NMR spectroscopy (see Table 4).

SUMMARY OF THE INVENTION

[0014] The present invention provides prokaryotic glycosyltransferaseenzymes and nucleic acids that encode the enzymes. In one embodiment,the invention provides isolated and/or recombinant nucleic acidmolecules that include a polynucleotide sequence that encodes apolypeptide selected from the group consisting of:

[0015] a) a polypeptide having lipid A biosynthesis acyltransferaseactivity, wherein the polypeptide comprises an amino acid sequence thatis at least about 70% identical to an amino acid sequence encoded bynucleotides 350-1234 (ORF 2a) of the LOS biosynthesis locus of C. jejunistrain OH4384 as shown in SEQ ID NO:1;

[0016] b) a polypeptide having glycosyltransferase activity, wherein thepolypeptide comprises an amino acid sequence that is at least about 70%identical to an amino acid sequence encoded by nucleotides 1234-2487(ORF 3a) of the LOS biosynthesis locus of C. jejuni strain OH4384 asshown in SEQ ID NO:1;

[0017] c) a polypeptide having glycosyltransferase activity, wherein thepolypeptide comprises an amino acid sequence that is at least about 50%identical to an amino acid sequence encoded by nucleotides 2786-3952(ORF 4a) of the LOS biosynthesis locus of C. jejuni strain OH4384 asshown in SEQ ID NO:1 over a region at least about 100 amino acids inlength;

[0018] d) a polypeptide having β1,4-GalNAc transferase activity, whereinthe GalNAc transferase polypeptide has an amino acid sequence that is atleast about 77% identical to an amino acid sequence as set forth in SEQID NO:17 over a region at least about 50 amino acids in length;

[0019] e) a polypeptide having β1,3-galactosyltransferase activity,wherein the galactosyltransferase polypeptide has an amino acid sequencethat is at least about 75% identical to an amino acid sequence as setforth in SEQ ID NO:27 or SEQ ID NO:29 over a region at least about 50amino acids in length;

[0020] f) a polypeptide having either α2,3 sialyltransferase activity orboth α2,3- and α2,8 sialyltransferase activity, wherein the polypeptidehas an amino acid sequence that is at least about 66% identical over aregion at least about 60 amino acids in length to an amino acid sequenceas set forth in one or more of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 orSEQ ID NO:10;

[0021] g) a polypeptide having sialic acid synthase activity, whereinthe polypeptide comprises an amino acid sequence that is at least about70% identical to an amino acid sequence encoded by nucleotides 6924-7961of the LOS biosynthesis locus of C. jejuni strain OH4384 as shown in SEQID NO:1;

[0022] h) a polypeptide having sialic acid biosynthesis activity,wherein the polypeptide comprises an amino acid sequence that is atleast about 70% identical to an amino acid sequence encoded bynucleotides 8021-9076 of the LOS biosynthesis locus of C. jejuni strainOH4384 as shown in SEQ ID NO:1;

[0023] i) a polypeptide having CNP-sialic acid synthetase activity,wherein the polypeptide comprises an amino acid sequence that is atleast about 65% identical to an amino acid sequence encoded bynucleotides 9076-9738 of the LOS biosynthesis locus of C. jejuni strainOH4384 as shown in SEQ ID NO:1;

[0024] j) a polypeptide having acetyltransferase activity, wherein thepolypeptide comprises an amino acid sequence that is at least about 65%identical to an amino acid sequence-encoded by nucleotides 9729-10559 ofthe LOS biosynthesis locus of C. jejuni strain OH4384 as shown in SEQ IDNO:1; and

[0025] k) a polypeptide having glycosyltransferase activity, wherein thepolypeptide comprises an amino acid sequence that is at least about 65%identical to an amino acid sequence encoded by a reverse complement ofnucleotides 10557-11366 of the LOS biosynthesis locus of C. jejunistrain OH4384 as shown in SEQ ID NO:1.

[0026] In presently preferred embodiments, the invention provides anisolated nucleic acid molecule that includes a polynucleotide sequencethat encodes one or more polypeptides selected from the group consistingof: a) a sialyltransferase polypeptide that has both an α2,3sialyltransferase activity and an α2,8 sialyltransferase activity,wherein the sialyltransferase polypeptide has an amino acid sequencethat is at least about 76% identical to an amino acid sequence as setforth in SEQ ID NO:3 over a region at least about 60 amino acids inlength; b) a GalNAc transferase polypeptide that has a β1,4-GalNActransferase activity, wherein the GalNAc transferase polypeptide has anamino acid sequence that is at least about 75% identical to an aminoacid sequence as set forth in SEQ ID NO:17 over a region at least about50 amino acids in length; and c) a galactosyltransferase polypeptidethat has β1,3-galactosyltransferase activity, wherein thegalactosyltransferase polypeptide has an amino acid sequence that is atleast about 75% identical to an amino acid sequence as set forth in SEQID NO:27 over a region at least about 50 amino acids in length.

[0027] Also provided by the invention are expression cassettes andexpression vectors in which a glycosyltransferase nucleic acid of theinvention is operably linked to a promoter and other control sequencesthat facilitate expression of the glycosyltransferases in a desired hostcell. Recombinant host cells that express the glycosyltransferases ofthe invention are also provided.

[0028] The invention also provides isolated and/or recombinantlyproduced polypeptides selected from the group consisting of:

[0029] a) a polypeptide having lipid A biosynthesis acyltransferaseactivity, wherein the polypeptide comprises an amino acid sequence thatis at least about 70% identical to an amino acid sequence encoded bynucleotides 350-1234 (ORF 2a) of the LOS biosynthesis locus of C. jejunistrain OH4384 as shown in SEQ ID NO:1;

[0030] b) a polypeptide having glycosyltransferase activity, wherein thepolypeptide comprises an amino acid sequence that is at least about 70%identical to an amino acid sequence encoded by nucleotides 1234-2487(ORF 3a) of the LOS biosynthesis locus of C. jejuni strain OH4384 asshown in SEQ ID NO:1;

[0031] c) a polypeptide having glycosyltransferase activity, wherein thepolypeptide comprises an amino acid sequence that is at least about 50%identical to an amino acid sequence encoded by nucleotides 2786-3952(ORF 4a) of the LOS biosynthesis locus of C. jejuni strain OH4384 asshown in SEQ ID NO:1 over a region at least about 100 amino acids inlength;

[0032] d) a polypeptide having β1,4-GalNAc transferase activity, whereinthe GalNAc transferase polypeptide has an amino acid sequence that is atleast about 77% identical to an amino acid sequence as set forth in SEQID NO:17 over a region at least about 50 amino acids in length;

[0033] e) a polypeptide having o,3-galactosyltransferase activity,wherein the galactosyltransferase polypeptide has an amino acid sequencethat is at least about 75% identical to an amino acid sequence as setforth in SEQ ID NO:27 or SEQ ID NO:29 over a region at least about 50amino acids in length;

[0034] f) a polypeptide having either α2,3 sialyltransferase activity orboth α2,3 and α2,8 sialyltransferase activity, wherein the polypeptidehas an amino acid sequence that is at least about 66% identical to anamino acid sequence as set forth in SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7 or SEQ ID NO:10 over a region at least about 60 amino acids inlength;

[0035] g) a polypeptide having sialic acid synthase activity, whereinthe polypeptide comprises an amino acid sequence that is at least about70% identical to an amino acid sequence encoded by nucleotides 6924-7961of the LOS biosynthesis locus of C. jejuni strain OH4384 as shown in SEQID NO:1;

[0036] h) a polypeptide having sialic acid biosynthesis activity,wherein the polypeptide comprises an amino acid sequence that is atleast about 70% identical to an amino acid sequence encoded bynucleotides 8021-9076 of the LOS biosynthesis locus of C. jejuni strainOH4384 as shown in SEQ ID NO:1;

[0037] i) a polypeptide having CMP-sialic acid synthetase activitywherein the polypeptide comprises an amino acid sequence that is atleast about 65% identical to an amino acid sequence encoded bynucleotides 9076-9738 of the LOS biosynthesis locus of C. jejuni strainH011384 as shown in SEQ ID NO:1;

[0038] j) a polypeptide having acetyltransferase activity, wherein thepolypeptide comprises an amino acid sequence that is at least about 65%identical to an amino acid sequence encoded by nucleotides 9729-10559 ofthe LOS biosynthesis locus of C. jejuni strain OH4384 as shown in SEQ IDNO:1; and

[0039] k) a polypeptide having glycosyltransferase activity, wherein thepolypeptide comprises an amino acid sequence that is at least about 65%identical to an amino acid sequence encoded by a reverse complement ofnucleotides 10557-11366 of the LOS biosynthesis locus of C. jejunistrain OH4384 as shown in SEQ ID NO:1.

[0040] In presently preferred embodiments, the invention providesglycosyltransferase polypeptides including: a) a sialyltransferasepolypeptide that has both an α2,3 sialyltransferase activity and an α2.Ssialyltransferase activity, wherein the sialyltransferase polypeptidehas an amino acid sequence that is at least about 76% identical to anamino acid sequence as set forth in SEQ ID NO:3 over a region at leastabout 60 amino acids in length; b) a GalNAc transferase polypeptide thathas a β1,4-GalNAc transferase activity, wherein the GalNAc transferasepolypeptide has an amino acid sequence that is at least about 75%identical to an amino acid sequence as set forth in SEQ ID NO:17, over aregion at least about 50 amino acids in length; and c) agalactosyltransferase polypeptide that has β1,3-galactosyltransferaseactivity, wherein the galactosyltransferase polypeptide has an aminoacid sequence that is at least about 75% identical to an amino acidsequence as set forth in SEQ ID NO:27 or SEQ ID NO:29 over a region atleast about 50 amino acids in length.

[0041] The invention also provides reaction mixtures for the synthesisof a sialylated oligosaccharide. The reaction mixtures include asialyltransferase polypeptide which has both an α2,3 sialyltransferaseactivity and an α2,8 sialyltransferase activity. Also present in thereaction mixtures are a galactosylated acceptor moiety and asialyl-nucleotide sugar. The sialyltransferase transfers a first sialicacid residue from the sialyl-nucleotide sugar (e.g., CMP-sialic acid) tothe galactosylated acceptor moiety in an α2,3 linkage, and further addsa second sialic acid residue to the first sialic acid residue in an α2,8linkage.

[0042] In another embodiment, the invention provides methods forsynthesizing a sialylated oligosaccharide. These methods involveincubating a reaction mixture that includes a sialyltransferasepolypeptide which has both an α2,3 sialyltransferase activity and anα2,8 sialyltransferase activity, a galactosylated acceptor moiety, and asialyl-nucleotide sugar, under suitable conditions wherein thesialyltransferase polypeptide transfers a first sialic acid residue fromthe sialyl-nucleotide sugar to the galactosylated acceptor moiety in anα2,3 linkage, and further transfers a second sialic acid residue to thefirst sialic acid residue in an α2,8 linkage.

DETAILED DESCRIPTION

[0043] Definitions

[0044] The glycosyltransferases, reaction mixtures, and methods of theinvention are useful for transferring a monosaccharide from a donorsubstrate to an acceptor molecule. The addition generally takes place atthe non-reducing end of an oligosaccharide or carbohydrate moiety on abiomolecule. Biomolecules as defined here include, but are not limitedto, biologically significant molecules such as carbohydrates, proteins(e.g., glycoproteins), and lipids (e.g., glycolipids, phospholipids,sphingolipids and gangliosides).

[0045] The following abbreviations are used herein:

[0046] Ara=arabinosyl;

[0047] Fru=fructosyl;

[0048] Fuc=fucosyl;

[0049] Gal=galactosyl;

[0050] GalNAc=N-acetylgalactosaminyl;

[0051] Glc=glucosyl;

[0052] GlcNAc=N-acetylglucosaminyl;

[0053] Man=mannosyl; and

[0054] NeuAc=sialyl (N-acetylneuraminyl).

[0055] The term “sialic acid” refers to any member of a family ofnine-carbon carboxylated sugars. The most common member of the sialicacid family is N-acetyl-neuraminic acid(2-keto-5-acetamindo-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member ofthe family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which theN-acetyl group of NeuAc is hydroxylated. A third sialic acid familymember is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J.Biol. Chem. 261: 11550-11557; Kanarnon et al. (1990) J. Biol. Chem. 265:21811-21819. Also included are 9-substituted sialic acids such as a9-O-C₁-C₆ acyl-Neu5Ac like 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac,9-deoxy-9-fluoro-Neu5Ac and 9-azido-9-deoxy-Neu5Ac. For review of thesialic acid family, see, e.g., Varki (1992) Glycobiology 2: 2540; SialicAcids: Chemistry, Metabolism and Function, R. Schauer, Ed.(Springer-Verlag, New York (1992); Schauer, Methods in Enzymology, 50:64-89 (1987), and Schaur, Advances in Carbohydrate Chemistry andBiochemistry, 40: 131-234.The synthesis and use of sialic acid compoundsin a sialylation procedure is disclosed in international application WO92/16640, published Oct. 1, 1992.

[0056] Donor substrates for glycosyltransferases are activatednucleotide sugars. Such activated sugars generally consist of uridineand guanosine diphosphates, and cytidine monophosphate derivatives ofthe sugars in which the nucleoside diphosphate or monophosphate servesas a leaving group. Bacterial, plant, and fungal systems can sometimesuse other activated nucleotide sugars.

[0057] Oligosaccharides are considered to have a reducing end and anon-reducing end, whether or not the saccharide at the reducing end isin fact a reducing sugar. In accordance with accepted nomenclature,oligosaccharides are depicted herein with the non-reducing end on theleft and the reducing end on the right.

[0058] All oligosaccharides described herein are described with the nameor abbreviation for the non-reducing saccharide (e.g., Gal), followed bythe configuration of the glycosidic bond (α or β), the ring bond, thering position of the reducing saccharide involved in the bond, and thenthe name or abbreviation of the reducing saccharide (e.g., GlcNAc). Thelinkage between two sugars may be expressed, for example, as 2,3, 2→3,or (2,3). Each saccharide is a pyranose or furanose.

[0059] The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues of naturalnucleotides that hybridize to nucleic acids in manner similar tonaturally occurring nucleotides. Unless otherwise indicated, aparticular nucleic acid sequence includes the complementary sequencethereof.

[0060] The term “operably linked” refers to functional linkage between anucleic acid expression control sequence (such as a promoter, signalsequence, or array of transcription factor binding sites) and a secondnucleic acid sequence, wherein the expression control sequence affectstranscription and/or translation of the nucleic acid corresponding tothe second sequence.

[0061] A “heterologous polynucleotide” or a “heterologous nucleic acid”,as used herein, is one that originates from a source foreign to theparticular host cell, or, if from the same source, is modified from itsoriginal form. Thus, a heterologous glycosyltransferase gene in a hostcell includes a glycosyltransferase gene that is endogenous to theparticular host cell but has been modified. Modification of theheterologous sequence may occur, e.g., by treating the DNA with arestriction enzyme to generate a DNA fragment that is capable of beingoperably linked to a promoter. Techniques such as site-directedmutagenesis are also useful for modifying a heterologous sequence.

[0062] The term “recombinant” when used with reference to a cellindicates that the cell replicates a heterologous nucleic acid, orexpresses a peptide or protein encoded by a heterologous nucleic acid.Recombinant cells can contain genes that are not found within the native(non-recombinant) form of the cell. Recombinant cells also include thosethat contain genes that are found in the native form of the cell, butare modified and re-introduced into the cell by artificial means. Theterm also encompasses cells that contain a nucleic acid endogenous tothe cell that has been modified without removing the nucleic acid fromthe cell; such modifications include those obtained by gene replacement,site-specific mutation, and related techniques known to those of skillin the art.

[0063] A “recombinant nucleic acid” is a nucleic acid that is in a formthat is altered from its natural state. For example, the term“recombinant nucleic acid” includes a coding region that is operablylinked to a promoter and/or other expression control region, processingsignal, another coding region, and the like, to which the nucleic acidis not linked in its naturally occurring form. A “recombinant nucleicacid” also includes, for example, a coding region or other nucleic acidin which one or more nucleotides have been substituted, deleted,inserted, compared to the corresponding naturally occurring nucleicacid. The modifications include those introduced by in vitromanipulation, in vivo modification, synthesis methods, and the like.

[0064] A “recombinantly produced polypeptide” is a polypeptide that isencoded by a recombinant and/or heterologous nucleic acid. For example,a polypeptide that is expressed from a C. jejuniglycosyltransferase-encoding nucleic acid which is introduced into E.coli is a “recombinantly produced polypeptide.” A protein expressed froma nucleic acid that is operably linked to a non-native promoter is oneexample of a “recombinantly produced polypeptide. Recombinantly producedpolypeptides of the invention can be used to synthesize gangliosides andother oligosaccharides in their unpurified form (e.g., as a cell lysateor an intact cell), or after being completely or partially purified.

[0065] A “recombinant expression cassette” or simply an “expressioncassette” is a nucleic acid construct, generated recombinantly orsynthetically, with nucleic acid elements that are capable of affectingexpression of a structural gene in hosts compatible with such sequences.Expression cassettes include at least promoters and optionally,transcription termination signals. Typically, the recombinant expressioncassette includes a nucleic acid to be transcribed (e.g., a nucleic acidencoding a desired polypeptide), and a promoter. Additional factorsnecessary or helpful in effecting expression may also be used asdescribed herein. For example, an expression cassette can also includenucleotide sequences that encode a signal sequence that directssecretion of an expressed protein from the host cell. Transcriptiontermination signals, enhancers, and other nucleic acid sequences thatinfluence gene expression, can also be included in an expressioncassette.

[0066] A “subsequence” refers to a sequence of nucleic acids or aminoacids that comprise a part of a longer sequence of nucleic acids oramino acids (e.g., polypeptide) respectively.

[0067] The term “isolated” is meant to refer to material that issubstantially or essentially free from components which normallyaccompany the material as found in its native state. Typically, isolatedproteins or nucleic acids of the invention are at least about 80% pure,usually at least about 90%, and preferably at least about 95% pure.Purity or homogeneity can be indicated by a number of means well knownin the art, such as agarose or polyacrylarnide gel electrophoresis of aprotein or nucleic acid sample, followed by visualization upon staining.For certain purposes high resolution will be needed and HPLC or asimilar means for purification utilized. An “isolated” enzyme, forexample, is one which is substantially or essentially free fromcomponents which interfere with the activity of the enzyme. An “isolatednucleic acid” includes, for example, one that is not present in thechromosome of the cell in which the nucleic acid naturally occurs.

[0068] The terms “identical” or percent “identity,” in the context oftwo or more nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

[0069] The phrase “substantially identical,” in the context of twonucleic acids or polypeptides, refers to two or more sequences orsubsequences that have at least 60%, preferably 80%, most preferably90-95% nucleotide or amino acid residue identity, when compared andaligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms or by visual inspection.Preferably, the substantial identity exists over a region of thesequences that is at least about 50 residues in length, more preferablyover a region of at least about 100 residues, and most preferably thesequences are substantially identical over at least about 150 residues.In a most preferred embodiment, the sequences are substantiallyidentical over the entire length of the coding regions.

[0070] For sequence comparison, typically one sequence acts as areference sequence, to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

[0071] Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)(Ausubel)).

[0072] Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/). For example, the comparisonscan be performed using a BLASTN Version 2.0 algorithm with a wordlength(W) of 11, G=5, E=2, q=−2, and r=1, and a comparison of both strands.For amino acid sequences, the BLASTP Version 2.0 algorithm can be used,with the default values of wordlength (W) of 3, G=11, E=1, and aBLOSUM62 substitution matrix. (see Henikoff & Henikoff, Proc. Natl.Acad. Sci. USA 89:10915 (1989)).

[0073] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

[0074] The phrase “hybridizing specifically to”, refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. The term “stringentconditions” refers to conditions under which a probe will hybridize toits target subsequence, but to no other sequences. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target sequence hybridize to the targetsequence at equilibrium. (As the target sequences are generally presentin excess, at Tm, 50% of the probes are occupied at equilibrium).Typically, stringent conditions will be those in which the saltconcentration is less than about 1.0 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

[0075] A further indication that two nucleic acid sequences orpolypeptides are substantially identical is that the polypeptide encodedby the first nucleic acid is immunologically cross reactive with thepolypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

[0076] The phrases “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind preferentially to a particular protein and do not bind in asignificant amount to other proteins present in the sample. Specificbinding to a protein under such conditions requires an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane (1988)Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork, for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

[0077] “Conservatively modified variations” of a particularpolynucleotide sequence refers to those polynucleotides that encodeidentical or essentially identical amino acid sequences, or where thepolynucleotide does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of “conservatively modified variations.” Every polynucleotidesequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

[0078] Furthermore, one of skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (typically lessthan 5%, more typically less than 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. One of skill will appreciate that manyconservative variations of the fusion proteins and nucleic acid whichencode the fusion proteins yield essentially identical products. Forexample, due to the degeneracy of the genetic code, “silentsubstitutions” (i.e., substitutions of a nucleic acid sequence which donot result in an alteration in an encoded polypeptide) are an impliedfeature of every nucleic acid sequence which encodes an amino acid. Asdescribed herein, sequences are preferably optimized for expression in aparticular host cell used to produce the enzymes (e.g., yeast, human,and the like). Similarly, “conservative amino acid substitutions,” inone or a few amino acids in an amino acid sequence are substituted withdifferent amino acids with highly similar properties (see, thedefinitions section, supra), are also readily identified as being highlysimilar to a particular amino acid sequence, or to a particular nucleicacid sequence which encodes an amino acid. Such conservativelysubstituted variations of any particular sequence are a feature of thepresent invention. See also, Creighton (1984) Proteins, W. H. Freemanand Company. In addition, individual substitutions, deletions oradditions which alter, add or delete a single amino acid or a smallpercentage of amino acids in an encoded sequence are also“conservatively modified variations”.

[0079] Description of the Preferred Embodiments

[0080] The present invention provides novel glycosyltransferase enzymes,as well as other enzymes that are involved in enzyme-catalyzedoligosaccharide synthesis. The glycosyltransferases of the inventioninclude sialyltransferases, including a bifunctional sialyltransferasethat has both an α2,3 and an α2,8 sialyltransferase activity. Alsoprovided are β1,3-galactosyltransferases, β1,4-GalNAc transferases,sialic acid synthases, CMP-sialic acid synthetases, acetyltransferases,and other glycosyltransferases. The enzymes of the invention areprokaryotic enzymes, include those involved in the biosynthesis oflipooligosaccharides (LOS) in various strains of Campylobacter jejuni.The invention also provides nucleic acids that encode these enzymes, aswell as expression cassettes and expression vectors for use inexpressing the glycosyltransferases. In additional embodiments, theinvention provides reaction mixtures and methods in which one or more ofthe enzymes is used to synthesize an oligosaccharide.

[0081] The glycosyltransferases of the invention are useful for severalpurposes. For example, the glycosyltransferases are useful as tools forthe chemo-enzymatic syntheses of oligosaccharides, includinggangliosides and other oligosaccharides that have biological activity.The glycosyltransferases of the invention, and nucleic acids that encodethe glycosyltransferases, are also useful for studies of thepathogenesis mechanisms of organisms that synthesize ganglioside mimics,such as C. jejuni. The nucleic acids can be used as probes, for example,to study expression of the genes involved in ganglioside mimeticsynthesis. Antibodies raised against the glycosyltransferases are alsouseful for analyzing the expression patterns of these genes that areinvolved in pathogenesis. The nucleic acids are also useful fordesigning antisense oligonucleotides for inhibiting expression of theCampylobacter enzymes that are involved in the biosynthesis ofganglioside mimics that can mask the pathogens from the host's immunesystem.

[0082] The glycosyltransferases of the invention provide severaladvantages over previously available glycosyltransferases. Bacterialglycosyltransferases such as those of the invention can catalyze theformation of oligosaccharides that are identical to the correspondingmammalian structures. Moreover, bacterial enzymes are easier and lessexpensive to produce in quantity, compared to mammalianglycosyltransferases. Therefore, bacterial glycosyltransferases such asthose of the present invention are attractive replacements for mammalianglycosyltransferases, which can be difficult to obtain in large amounts.That the glycosyltransferases of the invention are of bacterial originfacilitates expression of large quantities of the enzymes usingrelatively inexpensive prokaryotic expression systems. Typically,prokaryotic systems for expression of polypeptide products involves amuch lower cost than expression of the polypeptides in mammalian cellculture systems.

[0083] Moreover, the novel bifunctional sialyltransferases of theinvention simplify the enzymatic synthesis of biologically importantmolecules, such as gangliosides, that have a sialic acid attached by anα2,8 linkage to a second sialic acid, which in turn is α2,3-linked to agalactosylated acceptor. While previous methods for synthesizing thesestructures required two separate sialyltransferases, only onesialyltransferase is required when the bifunctional sialyltransferase ofthe present invention is used. This avoids the costs associated withobtaining a second enzyme, and can also reduce the number of stepsinvolved in synthesizing these compounds.

[0084] A. Glycosyltransferases and Associated Enzymes

[0085] The present invention provides prokaryotic glycosyltransferasepolypeptides, as well as other enzymes that are involved in theglycosyltransferase-catalyzed synthesis of oligosaccharides, includinggangliosides and ganglioside mimics. In presently preferred embodiments,the polypeptides include those that are encoded by open reading frameswithin the lipooligosaccharide (LOS) locus of Campylobacter species(FIG. 1). Included among the enzymes of the invention areglycosyltransferases, such as sialyltransferases (including abifunctional sialyltransferase), β1,4-GalNAc transferases, andβ1,3-galactosyltransferases, among other enzymes as described herein.Also provided are accessory enzymes such as, for example, CMP-sialicacid synthetase, sialic acid synthase, acetyltransferase, anacyltransferase that is involved in lipid A biosynthesis, and an enzymeinvolved in sialic acid biosynthesis.

[0086] The glycosyltransferases and accessory polypeptides of theinvention can be purified from natural sources, e.g., prokaryotes suchas Campylobacter species. In presently preferred embodiments, theglycosyltransferases are obtained from C. jejuni, in particular from C.jejuni serotype O:19, including the strains OH4384 and OH4382. Alsoprovided are glycosyltransferases and accessory enzymes obtained from C.jejuni serotypes O:10, O:41, and O:2. Methods by which theglycosyltransferase polypeptides can be purified include standardprotein purification methods including, for example, ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982) Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)).

[0087] In presently preferred embodiments, the glycosyltransferase andaccessory enzyme polypeptides of the invention are obtained byrecombinant expression using the glycosyltransferase- and accessoryenzyme-encoding nucleic acids described herein. Expression vectors andmethods for producing the glycosyltransferases are described in detailbelow.

[0088] In some embodiments, the glycosyltransferase polypeptides areisolated from their natural milieu, whether recombinantly produced orpurified from their natural cells. Substantially pure compositions of atleast about 90 to 95% homogeneity are preferred for some applications,and 98 to 99% or more homogeneity are most preferred. Once purified,partially or to homogeneity as desired, the polypeptides may then beused (e.g., as immunogens for antibody production or for synthesis ofoligosaccharides, or other uses as described herein or apparent to thoseof skill in the art). The glycosyltransferases need not, however, beeven partially purified for use to synthesize a desired saccharidestructure. For example, the invention provides recombinantly producedenzymes that are expressed in a heterologous host cell and/or from arecombinant nucleic acid. Such enzymes of the invention can be used whenpresent in a cell lysate or an intact cell, as well as in purified form.

[0089] 1. Sialyltransferases

[0090] In some embodiments, the invention provides sialyltransferasepolypeptides. The sialyltransferases have an α2,3-sialyltransferaseactivity, and in some cases also have an α2,8 sialyltransferaseactivity. These bifunctional sialyltransferases, when placed in areaction mixture with a suitable saccharide acceptor (e.g., a saccharidehaving a terminal galactose) and a sialic acid donor (e.g., CMP-sialicacid) can catalyze the transfer of a first sialic acid from the donor tothe acceptor in an α2,3 linkage. The sialyltransferase then catalyzesthe transfer of a second sialic acid from a sialic acid donor to thefirst sialic acid residue in an α2,8 linkage. This type ofSiaα2,8-Siaα2,3-Gal structure is often found in gangliosides, includingGD3 and GT1a as shown in FIG. 4.

[0091] Examples of bifunctional sialyltransferases of the invention arethose that are found in Campylobacter species, such as C jejuni. Apresently preferred bifunctional sialyltransferase of the invention isthat of the C. jejuni serotype O:19. One example of a bifunctionalsialyltransferase is that of C. jejuni strain OH 4384; thissialyltransferase has an amino acid sequence as shown in SEQ ID NO:3.Other bifunctional sialyltransferases of the invention generally have anamino acid sequence that is at least about 76% identical to the aminoacid sequence of the C. jejuni OH4384 bifunctional sialyltransferaseover a region at least about 60 amino acids in length. More preferably,the sialyltransferases of the invention are at least about 85% identicalto the OH 4384 sialyltransferase amino acid sequence, and still morepreferably at least about 95% identical to the amino acid sequence ofSEQ ID NO:3, over a region of at least 60 amino acids in length. Inpresently preferred embodiments, the region of percent identity extendsover a region longer than 60 amino acids. For example, in more preferredembodiments, the region of similarity extends over a region of at leastabout 100 amino acids in length, more preferably a region of at leastabout 150 amino acids in length, and most preferably over the fulllength of the sialyltransferase. Accordingly, the bifunctionalsialyltransferases of the invention include polypeptides that haveeither or both the α2,3- and α2,8-sialyltransferase activity and are atleast about 65% identical, more preferably at least about 70% identical,more preferably at least about 80% identical, and most preferably atleast about 90% identical to the amino acid sequence of the C. jejuni OH4384 CstII sialyltransferase (SEQ ID NO:3) over a region of thepolypeptide that is required to retain the respective sialyltransferaseactivities. In some embodiments, the bifunctional sialyltransferases ofthe invention are identical to C. jejuni OH 4384 CstII sialyltransferaseover the entire length of the sialyltransferase.

[0092] The invention also provides sialyltransferases that have α2,3sialyltransferase activity, but little or no α2,8 sialyltransferaseactivity. For example, CstII sialyltransferase of the C. jejuni O:19serostrain (SEQ ID NO:9) differs from that of strain OH 4384 by eightamino acids, but nevertheless substantially lacks α2,8 sialyltransferaseactivity (FIG. 3). The corresponding sialyltransferase from the O:2serotype strain NCTC 11168 (SEQ ID NO:10) is 52% identical to that ofOH4384, and also has little or no α2,8-sialyltranfserase activity.Sialyltransferases that are substantially identical to the CstIIsialyltransferase of C. jejuni strain O:10 (SEQ ID NO:5) and 0:41 (SEQID NO:7) are also provided. The sialyltransferases of the inventioninclude those that are at least about 65% identical, more preferably atleast about 70% identical, more preferably at least about 80% identical,and most preferably at least about 90% identical to the amino acidsequences of the C. jejuni O:10 (SEQ ID NO:5), O:41 (SEQ ID NO:7), O:19serostrain (SEQ ID NO:9), or O:2 serotype strain NCTC 11168 (SEQ IDNO:10). The sialyltransferases of the invention, in some embodiments,have an amino acid sequence that is identical to that of the O:10, O:41,O:19 serostrain or NCTC 11168 C. jejuni strains.

[0093] The percent identities can be determined by inspection, forexample, or can be determined using an alignment algorithm such as theBLASTP Version 2.0 algorithm using the default parameters, such as awordlength (W) of 3, G=11, E=1, and a BLOSUM62 substitution matrix.

[0094] Sialyltransferases of the invention can be identified, not onlyby sequence comparison, but also by preparing antibodies against the C.jejuni OH4384 bifunctional sialyltransferase, or othersialyltransferases provided herein, and determining whether theantibodies are specifically immunoreactive with a sialyltransferase ofinterest. To obtain a bifunctional sialyltransferase in particular, onecan identify an organism that is likely to produce a bifunctionalsialyltransferase by determining whether the organism displays both α2,3and α2,8-sialic acid linkages on its cell surfaces. Alternatively, or inaddition, one can simply do enzyme assays of an isolatedsialyltransferase to determine whether both sialyltransferase activitiesare present. 2. β1,4-GalNAc transferase

[0095] The invention also provides β1,4-GalNAc transferase polypeptides(e.g., CgtA). The β1,4-GalNAc transferases of the invention, when placedin a reaction mixture, catalyze the transfer of a GalNAc residue from adonor (e.g., UDP-GalNAc) to a suitable acceptor saccharide (typically asaccharide that has a terminal galactose residue). The resultingstructure, GalNAcβ1,4-Gal-, is often found in gangliosides and othersphingoids, among many other saccharide compounds. For example, the CgtAtransferase can catalyze the conversion of the ganglioside GM3 to GM2(FIG. 4).

[0096] Examples of the β1,4-GalNAc transferases of the invention arethose that are produced by Campylobacter species, such as C. jejuni. Oneexample of a β1,4-GalNAc transferase polypeptide is that of C. jejunistrain OH4384, which has an amino acid sequence as shown in SEQ IDNO:17. The β1,4-GalNAc transferases of the invention generally includean amino acid sequence that is at least about 75% identical to an aminoacid sequence as set forth in SEQ ID NO:17 over a region at least about50 amino acids in length. More preferably, the β1,4-GalNAc transferasesof the invention are at least about 85% identical to this amino acidsequence, and still more preferably are at least about 95% identical tothe amino acid sequence of SEQ ID NO:17, over a region of at least 50amino acids in length. In presently preferred embodiments, the region ofpercent identity extends over a longer region than 50 amino acids, morepreferably over a region of at least about 100 amino acids, and mostpreferably over the full length of the GalNAc transferase. Accordingly,the β1,4-GalNAc transferases of the invention include polypeptides thathave β1,4-GalNAc transferase activity and are at least about 65%identical, more preferably at least about 70% identical, more preferablyat least about 80% identical, and most preferably at least about 90%identical to the amino acid sequence of the C. jejuni OH 4384β1,4-GalNAc transferases (SEQ ID NO:17) over a region of the polypeptidethat is required to retain the β1,4-GalNAc transferase activity. In someembodiments, the β1,4-GalNAc transferases of the invention are identicalto C. jejuni OH 4384 β1,4-GalNAc transferase over the entire length ofthe β1,4-GalNAc transferase.

[0097] Again, the percent identities can be determined by inspection,for example, or can be determined using an alignment algorithm such asthe BLASTP Version 2.0 algorithm with a wordlength (W) of 3, G=11, E=1,and a BLOSUM62 substitution matrix.

[0098] One can also identify β1,4-GalNAc transferases of the inventionby immunoreactivity. For example, one can prepare antibodies against theC. jejuni OH4384 β1,4-GalNAc transferase of SEQ ID NO:17 and determinewhether the antibodies are specifically immunoreactive with aβ1,4-GalNAc transferase of interest.

[0099] 3. β1,3-Galactosyltransferases

[0100] Also provided by the invention are β1,3-galactosyltransferases(CgtB). When placed in a suitable reaction medium, theβ1,3-galactosyltransferases of the invention catalyze the transfer of agalactose residue from a donor (e.g., UDP-Gal) to a suitable saccharideacceptor (e.g., saccharides having a terminal GalNAc residue). Among thereactions catalyzed by the β1,3-galactosyltransferases is the transferof a galactose residue to the oligosaccharide moiety of GM2 to form theGM1a oligosaccharide moiety.

[0101] Examples of the β1,3-galactosyltransferases of the invention arethose produced by Campylobacter species, such as C. jejuni. For example,one β1,3-galactosyl-transferase of the invention is that of C. jejunistrain OH4384, which has the amino acid sequence shown in SEQ ID NO:27.

[0102] Another example of a β1,3-galactosyltransferase of the inventionis that of the C. jejuni O:2 serotype strain NCTC 11168. The amino acidsequence of this galactosyltransferase is set forth in SEQ ID NO:29.This galactosyltransferase expresses well in E. coli, for example, andexhibits a high amount of soluble activity. Moreover, unlike the OH4384CgtB, which can add more than one galactose if a reaction mixturecontains an excess of donor and is incubated for a sufficiently longperiod of time, the NCTC 11168 β1,3-galactose does not have asignificant amount of polygalactosyltransferase activity. For someapplications, the polygalactosyltransferase activity of the OH4384enzyme is desirable, but in other applications such as synthesis of GM1mimics, addition of only one terminal galactose is desirable.

[0103] The β1,3-galactosyltransferases of the invention generally havean amino acid sequence that is at least about 75% identical to an aminoacid sequence of the OH 4384 or NCTC 11168.CgtB as set forth in SEQ IDNO:27 and SEQ ID NO:29, respectively, over a region at least about 50amino acids in length. More preferably, the β1,3-galactosyltransferasesof the invention are at least about 85% identical to either of theseamino acid sequences, and still more preferably are at least about 95%identical to the amino acid sequences of SEQ ID NO:27 or SEQ ID NO:29,over a region of at least 50 amino acids in length. In presentlypreferred embodiments, the region of percent identity extends over alonger region than 50 amino acids, more preferably over a region of atleast about 100 amino acids, and most preferably over the full length ofthe galactosyltransferase. Accordingly, the β1,3-galactosyltransferasesof the invention include polypeptides that haveβ1,3-galactosyltransferase activity and are at least about 65%identical, more preferably at least about 70% identical, more preferablyat least about 80% identical, and most preferably at least about 90%identical to the amino acid sequence of the C. jejuni OH4384β1,3-galactosyltransferase (SEQ ID NO:27) or the NCTC 11168galactosyltransferase (SEQ ID NO:29) over a region of the polypeptidethat is required to retain the β1,3-galactosyltransferase activity. Insome embodiments, the β1,3-galactosyltransferase of the invention areidentical to C. jejuni OH 4384 or NCTC 11168 β1,3-galactosyltransferaseover the entire length of the β1,3-galactosyltransferase.

[0104] The percent identities can be determined by inspection, forexample, or can be determined using an alignment algorithm such as theBLASTP Version 2.0 algorithm with a wordlength (W) of 3, G=11, E=1, anda BLOSUM62 substitution matrix.

[0105] The β1,3-galactosyltransferases of the invention can be obtainedfrom the respective Campylobacter species, or can be producedrecombinantly. One can identify the glycosyltransferases by assays ofenzymatic activity, for example, or by detecting specificimmunoreactivity with antibodies raised against the C. jejuni OH4384β1,3-galactosyltransferase having an amino acid sequence as set forth inSEQ ID NO:27 or the C. jejuni NCTC 11168 β1,3 galactosyltransferase asset forth in SEQ ID NO:29.

[0106] 4. Additional Enzymes Involved in LOS Biosynthetic Pathway

[0107] The present invention also provides additional enzymes that areinvolved in the biosynthesis of oligosaccharides such as those found onbacterial lipooligosaccharides. For example, enzymes involved in thesynthesis of CMP-sialic acid, the donor for sialyltransferases, areprovided. A sialic acid synthase is encoded by open reading frame (ORF)8a of C. jejuni strain OH 4384 (SEQ ID NO:35) and by open reading frame8b of strain NCTC 11168 (see, Table 3). Another enzyme involved insialic acid synthesis is encoded by ORF 9a of OH 4384 (SEQ ID NO:36) and9b of NCTC 11168. A CMP-sialic acid synthetase is encoded by ORF 10a(SEQ ID NO:37) and 10b of OH 4384 and NCTC 11168, respectively.

[0108] The invention also provides an acyltransferase that is involvedin lipid A biosynthesis. This enzyme is encoded by open reading frame 2aof C. jejuni strain OH4384 (SEQ ID NO:32) and by open reading frame 2Bof strain NCTC 11168. An acetyltransferase is also provided; this enzymeis encoded by ORF 11a of strain OH 4384 (SEQ ID NO:38); no homolog isfound in the LOS biosynthesis locus of strain NCTC 11168.

[0109] Also provided are three additional glycosyltransferases. Theseenzymes are encoded by ORFs 3a (SEQ ID NO:33), 4a (SEQ ID NO:34), and12a (SEQ ID NO:39) of strain OH 4384 and ORFs 3b, 4b, and 12b of strainNCTC 11168.

[0110] The invention includes, for each of these enzymes, polypeptidesthat-include an an amino acid sequence that is at least about 75%identical to an amino acid sequence as set forth herein over a region atleast about 50 amino acids in length. More preferably, the enzymes ofthe invention are at least about 85% identical to the respective aminoacid sequence, and still more preferably are at least about 95%identical to the amino acid sequence, over a region of at least 50 aminoacids in length. In presently preferred embodiments, the region ofpercent identity extends over a longer region than 50 amino acids, morepreferably over a region of at least about 100 amino acids, and mostpreferably over the full length of the enzyme. Accordingly, the enzymesof the invention include polypeptides that have the respective activityand are at least about 65% identical, more preferably at least about 70%identical, more preferably at least about 80% identical, and mostpreferably at least about 90% identical to the amino acid sequence ofthe corresponding enzyme as set forth herein over a region of thepolypeptide that is required to retain the respective enzymaticactivity. In some embodiments, the enzymes of the invention areidentical to the corresponding C. jejuni OH 4384 enzymes over the entirelength of the enzyme.

[0111] B. Nucleic Acids that Encode Glycosyltransferases and RelatedEnzymes

[0112] The present invention also provides isolated and/or recombinantnucleic acids that encode the glycosyltransferases and other enzymes ofthe invention. The glycosyltransferase-encoding nucleic acids of theinvention are useful for several purposes, including the recombinantexpression of the corresponding glycosyltransferase polypeptides, and asprobes to identify nucleic acids that encode other glycosyltransferasesand to study regulation and expression of the enzymes.

[0113] Nucleic acids of the invention include those that encode anentire glycosyltransferase enzyme such as those described above, as wellas those that encode a subsequence of a glycosyltransferase polypeptide.For example, the invention includes nucleic acids that encode apolypeptide which is not a full-length glycosyltransferase enzyme, butnonetheless has glycosyltransferase activity. The nucleotide sequencesof the LOS locus of C. jejuni strain OH4384 is provided herein as SEQ IDNO:1, and the respective reading frames are identified. Additionalnucleotide sequences are also provided, as discussed below. Theinvention includes not only nucleic acids that include the nucleotidesequences as set forth herein, but also nucleic acids that aresubstantially identical to, or substantially complementary to, theexemplified embodiments. For example, the invention includes nucleicacids that include a nucleotide sequence that is at least about 70%identical to one that is set forth herein, more preferably at least 75%,still more preferably at least 80%, more preferably at least 85%, stillmore preferably at least 90%, and even more preferably at least about95% identical to an exemplified nucleotide sequence. The region ofidentity extends over at least about 50 nucleotides, more preferablyover at least about 100 nucleotides, still more preferably over at leastabout 500 nucleotides. The region of a specified percent identity, insome embodiments, encompasses the coding region of a sufficient portionof the encoded enzyme to retain the respective enzyme activity. Thespecified percent identity, in preferred embodiments, extends over thefull length of the coding region of the enzyme.

[0114] The nucleic acids that encode the glycosyltransferases of theinvention can be obtained using methods that are known to those of skillin the art. Suitable nucleic acids (e.g., cDNA, genomic, or subsequences(probes)) can be cloned, or amplified by in vitro methods such as thepolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (SSR). A wide variety of cloning and invitro amplification methodologies are well-known to persons of skill.Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152Academic Press, Inc., San Diego, Calif. (Berger); Sanbrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook et al.);Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashionet al., U.S. Pat. No. 5,017,478; and Carr, European Patent No.0,246,864. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al., eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C & EN 36-47; The Journal Of NIH Research (1991) 3:81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelliet al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lornell et al. (1989)J. Clin. Chem., 35: 1826; Landegren et al., (1988) Science 241:1077-1080; Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace(1989) Gene 4: 560; and Barringer et al. (1990) Gene 89: 117. Improvedmethods of cloning in vitro amplified nucleic acids are described inWallace et al., U.S. Pat. No. 5,426,039.

[0115] Nucleic acids that encode the glycosyltransferase polypeptides ofthe invention, or subsequences of these nucleic acids, can be preparedby any suitable method as described above, including, for example,cloning and restriction of appropriate sequences. As an example, one canobtain a nucleic-acid that encodes a glycosyltransferase of theinvention by routine cloning methods. A known nucleotide sequence of agene that encodes the glycosyltransferase of interest, such as aredescribed herein, can be used to provide probes that specificallyhybridize to a gene that encodes a suitable enzyme in a genomic DNAsample, or to a mRNA in a total RNA sample (e.g., in a Southern orNorthern blot). Preferably, the samples are obtained from prokaryoticorganisms, such as Campylobacter species. Examples of Campylobacterspecies of particular interest include C. jejuni. Many C. jejuni O:19strains synthesize ganglioside mimics and are useful as a source of theglycosyltransferases of the invention.

[0116] Once the target glycosyltransferase nucleic acid is identified,it can be isolated according to standard methods known to those of skillin the art (see, e.g., Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, 2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory;Berger and Kimmel (1987) Methods in Enzymology, Vol. 152: Guide toMolecular Cloning Techniques, San Diego: Academic Press, Inc.; orAusubel et al. (1987) Current Protocols in Molecular Biology, GreenePublishing and Wiley-Interscience, New York).

[0117] A nucleic acid that encodes a glycosyltransferase of theinvention can also be cloned by detecting its expressed product by meansof assays based on the physical, chemical, or immunological properties.For example, one can identify a cloned bifunctionalsialyltransferase-encoding nucleic acid by the ability of a polypeptideencoded by the nucleic acid to catalyze the coupling of a sialic acid inan α2,3-linkage to a galactosylated acceptor, followed by the couplingof a second sialic acid residue to the first sialic acid in an α2,8linkage. Similarly, one can identify a cloned nucleic acid that encodesa β1,4-GalNAc transferase or a β1,3-galactosyltransferase by the abilityof the encoded polypeptide to catalyze the transfer of a GalNAc residuefrom UDP-GalNAc, or a galactose residue from UDP-Gal, respectively, to asuitable acceptor. Suitable assay conditions are known in the art, andinclude those that are described in the Examples. Other physicalproperties of a polypeptide expressed from a particular nucleic acid canbe compared to properties of known glycosyltransferase polypeptides ofthe invention, such as those described herein, to provide another methodof identifying nucleic acids that encode glycosyltransferases of theinvention. Alternatively, a putative glycosyltransferase gene can bemutated; and its role as a glycosyltransferase established by detectinga variation in the ability to produce the respective glycoconjugate.

[0118] In other embodiments, glycosyltransferase-encoding nucleic acidscan be cloned using DNA amplification methods such as polymerase chainreaction (PCR). Thus, for example, the nucleic acid sequence orsubsequence is PCR amplified, preferably using a sense primer containingone restriction site (e.g., XbaI) and an antisense primer containinganother restriction site (e.g., HindIII). This will produce a nucleicacid encoding the desired glycosyltransferase amino acid sequence orsubsequence and having terminal restriction sites. This nucleic acid canthen be easily ligated into a vector containing a nucleic acid encodingthe second molecule and having the appropriate corresponding restrictionsites. Suitable PCR primers can be determined by one of skill in the artusing the sequence information provided herein. Appropriate restrictionsites can also be added to the nucleic acid encoding theglycosyltransferase of the invention, or amino acid subsequence, bysite-directed mutagenesis. The plasmid containing theglycosyltransferase-encoding nucleotide sequence or subsequence iscleaved with the appropriate restriction endonuclease and then ligatedinto an appropriate vector for amplification and/or expression accordingto standard methods.

[0119] Examples of suitable primers suitable for amplification of theglycosyltransferase-encoding nucleic acids of the invention are shown inTable 2; some of the primer pairs are designed to provide a 5′ NdeIrestriction site and a 3′ SalI site on the amplified fragment. Theplasmid containing the enzyme-encoding sequence or subsequence iscleaved with the appropriate restriction endonuclease and then ligatedinto an appropriate vector for amplification and/or expression accordingto standard methods.

[0120] As an alternative to cloning a glycosyltransferase-encodingnucleic acid, a suitable nucleic acid can be chemically synthesized froma known sequence that encodes a glycosyltransferase of the invention.Direct chemical synthesis methods include, for example, thephosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99;the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al. (1981)Tetra. Lett., 22: 1859-1862; and the solid support method of U.S. Pat.No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is often limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences. Alternatively, subsequences may be clonedand the appropriate subsequences cleaved using appropriate restrictionenzymes. The fragments can then be ligated to produce the desired DNAsequence.

[0121] In some embodiments, it may be desirable to modify theenzyme-encoding nucleic acids. One of skill will recognize many ways ofgenerating alterations in a given nucleic acid construct. Suchwell-known methods include site-directed mutagenesis, PCR amplificationusing degenerate oligonucleotides, exposure of cells containing thenucleic acid to mutagenic agents or radiation, chemical synthesis of adesired oligonucleotide (e.g., in conjunction with ligation and/orcloning to generate large nucleic acids) and other well-knowntechniques. See, e.g., Giliman and Smith (1979) Gene 8:81-97, Roberts etal. (1987) Nature 328: 731-734.

[0122] In a presently preferred embodiment, the recombinant nucleicacids present in the cells of the invention are modified to providepreferred codons which enhance translation of the nucleic acid in aselected organism (e.g., E. coli preferred codons are substituted into acoding nucleic acid for expression in E. coli).

[0123] The present invention includes nucleic acids that are isolated(i.e., not in their native chromosomal location) and/or recombinant(i.e., modified from their original form, present in a non-nativeorganism, etc.).

[0124] 1. Sialyltransferases

[0125] The invention provides nucleic acids that encodesialyltransferases such as those described above. In some embodiments,the nucleic acids of the invention encode bifunctional sialyltransferasepolypeptides that have both an α2,3 sialyltransferase activity and anα2,8 sialyltransferase activity. These sialyltransferase nucleic acidsencode a sialyltransferase polypeptide that has an amino acid sequencethat is at least about 76% identical to an amino acid sequence as setforth in SEQ ID NO:3 over a region at least about 60 amino acids inlength. More preferably the sialyltransferases encoded by the nucleicacids of the invention are at least about 85% identical to the aminoacid sequence of SEQ ID NO:3, and still more preferably at least about95% identical to the amino acid sequence of SEQ ID NO:3, over a regionof at least 60 amino acids in length. In presently preferredembodiments, the region of percent identity extends over a longer regionthan 60 amino acids, more preferably over a region of at least about 100amino acids, and most preferably over the full length of thesialyltransferase. In a presently preferred embodiment, thesialyltransferase-encoding nucleic acids of the invention encode apolypeptide having the amino acid sequence as shown in SEQ ID NO:3.

[0126] An example of a nucleic acid of the invention is an isolatedand/or recombinant form of a bifunctional sialyltransferase-encodingnucleic acid of C. jejuni OH4384. The nucleotide sequence of thisnucleic acid is shown in SEQ ID NO:2. The sialyltransferase-encodingpolynucleotide sequences of the invention are typically at least about75% identical to the nucleic acid sequence of SEQ ID NO:2 over a regionat least about 50 nucleotides in length. More preferably, thesialyltransferase-encoding nucleic acids of the invention are at leastabout 85% identical to this nucleotide sequence, and still morepreferably are at least about 95% identical to the nucleotide sequenceof SEQ ID NO:2, over a region of at least 50 amino acids in length. Inpresently preferred embodiments, the region of the specified percentidentity threshold extends over a longer region than 50 nueleotides,more preferably over a region of at least about 100 nucleotides, andmost preferably over the full length of the sialyltransferase-encodingregion. Accordingly, the invention provides bifunctionalsialyltransferase-encoding nucleic acids that are substantiallyidentical to that of the C. jejuni strain OH4384 cstII as set forth inSEQ ID NO:2 or strain O:10 (SEQ ID NO:4).

[0127] Other sialyltransferase-encoding nucleic acids of the inventionencode sialyltransferases have α2,3 sialyltransferase activity but lacksubstantial α2,8 sialyltransferase activity. For example, nucleic acidsthat encode a CstII α2,3 sialyltransferase from C. jejuni serostrainO:19 (SEQ ID NO:8) and NCTC 11168 are provided by the invention; theseenzymes have little or no α2,8-sialyltransferase activity (Table 6).

[0128] To identify nucleic acids of the invention, one can use visualinspection, or can use a suitable alignment algorithm. An alternativemethod by which one can identify a bifunctionalsialyltransferase-encoding nucleic acid of the invention is byhybridizing, under stringent conditions, the nucleic acid of interest toa nucleic acid that includes a polynucleotide sequence of asialyltransferase as set forth herein.

[0129] 2. β1,4-GalNAc transferases

[0130] Also provided by the invention are nucleic acids that includepolynucleotide sequences that encode a GalNAc transferase polypeptidethat has a β1,4-GalNAc transferase activity. The polynucleotidesequences encode a GalNAc transferase polypeptide that has an amino acidsequence that is at least about 70% identical to the C. jejuni OH4384β1,4-GalNAc transferase, which has an amino acid sequence as set forthin SEQ ID NO:17, over a region at least about 50 amino acids in length.More preferably the GalNAc transferase polypeptide encoded by thenucleic acids of the invention are at least about 80% identical to thisamino acid sequence, and still more preferably at least about 90%identical to the amino acid sequence of SEQ ID NO:17, over a region ofat least 50 amino acids in length. In presently preferred embodiments,the region of percent identity extends over a longer region than 50amino acids, more preferably over a region of at least about 100 aminoacids, and most preferably over the full length of the GalNActransferase polypeptide. In a presently preferred embodiment, the GalNActransferase polypeptide-encoding nucleic acids of the invention encode apolypeptide having the amino acid sequence as shown in SEQ ID NO:17. Toidentify nucleic acids of the invention, one can use visual inspection,or can use a suitable alignment algorithm.

[0131] One example of a GalNAc transferase-encoding nucleic acid of theinvention is an isolated and/or recombinant form of the GalNActransferase-encoding nucleic acid of C. jejuni OH4384. This nucleic acidhas a nucleotide sequence as shown in SEQ ID NO:16. The GalNActransferase-encoding polynucleotide sequences of the invention aretypically at least about 75% identical to the nucleic acid sequence ofSEQ ID NO:16 over a region at least about 50 nucleotides in length. Morepreferably, the GalNAc transferase-encoding nucleic acids of theinvention are at least about 85% identical to this nucleotide sequence,and still more preferably are at least about 95% identical to thenucleotide sequence of SEQ ID NO:16, over a region of at least 50 aminoacids in length. In presently preferred embodiments, the region ofpercent identity extends over a longer region than 50 nucleotides, morepreferably over a region of at least about 100 nucleotides, and mostpreferably over the full length of the GalNAc transferase-encodingregion.

[0132] To identify nucleic acids of the invention, one can use visualinspection, or can use a suitable alignment algorithm. An alternativemethod-by which one can identify a GalNAc transferase-encoding nucleicacid of the invention is by hybridizing, under stringent conditions, thenucleic acid of interest to a nucleic acid that includes apolynucleotide sequence of SEQ ID NO:16.

[0133] 3. β1,3-Galactosyltransferases

[0134] The invention also provides nucleic acids that includepolynucleotide sequences that encode a polypeptide that hasβ1,3-galactosyltransferase activity (CgtB). Theβ1,3-galactosyltransferase polypeptides encoded by these nucleic acidsof the invention preferably include an amino acid sequence that is atleast about 75% identical to an amino acid sequence of a C. jejunistrain OH4384 β1,3-galactosyltransferase as set forth in SEQ ID NO:27,or to that of a strain NCTC 11168 β1,3-galactosyltransferase as setforth in SEQ ID NO:29, over a region at least about 50 amino acids inlength. More preferably, the galactosyltransferase polypeptides encodedby these nucleic acids of the invention are at least about 85% identicalto this amino acid sequence, and still more preferably are at leastabout 95% identical to the amino acid sequence of SEQ ID NO:27 or SEQ IDNO:29, over a region of at least 50 amino acids in length. In presentlypreferred embodiments, the region of percent identity extends over alonger region than 50 amino acids, more preferably over a region of atleast about 100 amino acids, and most preferably over the full length ofthe galactosyltransferase polypeptide-encoding region.

[0135] One example of a β1,3-galactosyltransferase-encoding nucleic acidof the invention is an isolated and/or recombinant form of theβ1,3-galactosyltransferase-encoding nucleic acid of C. jejuni OH4384.This nucleic acid includes a nucleotide sequence as shown in SEQ IDNO:26. Another suitable β1,3-galactosyltransferase-encoding nucleic acidincludes a nucleotide sequence of a C. jejuni NCTC 11168 strain, forwhich the nucleotide sequence is shown in SEQ ID NO:28. Theβ1,3-galactosyltransferase-encoding polynucleotide sequences of theinvention are typically at least about 75% identical to the nucleic acidsequence of SEQ ID NO:26 or that of SEQ ID NO:28 over a region at leastabout 50 nucleotides in length. More preferably, theβ1,3-galactosyltransferase-encoding nucleic acids of the invention areat least about 85% identical to at least one of these nucleotidesequences, and still more preferably are at least about 95% identical tothe nucleotide sequences of SEQ ID NO:26 and/or SEQ ID NO:28, over aregion of at least 50 amino acids in length. In presently preferredembodiments, the region of percent identity extends over a longer regionthan 50 nucleotides, more preferably over a region of at least about 100nucleotides, and most preferably over the full length of theβ1,3-galactosyltransferase-encoding region.

[0136] To identify nucleic acids of the invention, one can use visualinspection, or can use a suitable alignment algorithm. An alternativemethod by which one can identify a galactosyltransferasepolypeptide-encoding nucleic acid of the invention is by hybridizing,under stringent conditions, the nucleic acid of interest to a nucleicacid that includes a polynucleotide sequence of SEQ ID NO:26 or SEQ IDNO:28.

[0137] 4. Additional Enzymes Involved in LOS Biosynthetic Pathway

[0138] Also provided are nucleic acids that encode other enzymes thatare involved in the LOS biosynthetic pathway of prokaryotes such asCampylobacter. These nucleic acids encode enzymes such as, for example,sialic acid synthase, which is encoded by open reading frame (ORF) 8a ofC. jejuni strain OH 4384 and by open reading frame 8b of strain NCTC11168 (see, Table 3), another enzyme involved in sialic acid synthesis,which is encoded by ORF 9a of OH 4384 and 9b of NCTC 11168, and aCMP-sialic acid synthetase which is encoded by ORF 10a and 10b of OH4384 and NCTC 11168, respectively.

[0139] The invention also provides nucleic acids that encode anacyltransferase that is involved in lipid A biosynthesis. This enzyme isencoded by open reading frame 2a of C. jejuni strain OH4384 and by openreading frame 2B of strain NCTC 11168. Nucleic acids that encode anacetyltransferase are also provided; this enzyme is encoded by ORF 11aof strain OH 4384; no homolog is found in the LOS biosynthesis locus ofstrain NCTC 11168.

[0140] Also provided are nucleic acids that encode three additionalglycosyltransferases. These enzymes are encoded by ORFs 3a, 4a, and 12aof strain OH 4384 and ORFs 3b, 4b, and 12b of strain NH 11168 (FIG. 1).

[0141] C. Expression Cassettes and Expression of theGlycosyltransferases

[0142] The present invention also provides expression cassettes,expression vectors, and recombinant host cells that can be used toproduce the glycosyltransferases and other enzymes of the invention. Atypical expression cassette contains a promoter operably linked to anucleic acid that encodes the glycosyltransferase or other enzyme ofinterest. The expression cassettes are typically included on expressionvectors that are introduced into suitable host cells, preferablyprokaryotic host cells. More than one glycosyltransferase polypeptidecan be expressed in a single host cell by placing multipletranscriptional cassettes in a single expression vector, by constructinga gene that encodes a fusion protein consisting of more than oneglycosyltransferase, or by utilizing different expression vectors foreach glycosyltransferase.

[0143] In a preferred embodiment, the expression cassettes are usefulfor expression of the glycosyltransferases in prokaryotic host cells.Commonly used prokaryotic control sequences, which are defined herein toinclude promoters for transcription initiation, optionally with anoperator, along with ribosome binding site sequences, include suchcommonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056),the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad.Sci. U.S.A. (1983) 80:21-25); and the lambda-derived P_(L) promoter andN-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128).The particular promoter system is not critical to the invention, anyavailable promoter that functions in prokaryotes can be used.

[0144] Either constitutive or regulated promoters can be used in thepresent invention. Regulated promoters can be advantageous because thehost cells can be grown to high densities before expression of theglycosyltransferase polypeptides is induced. High level expression ofheterologous proteins slows cell growth in some situations. Regulatedpromoters especially suitable for use in E. coli include thebacteriophage lambda P_(L) promoter, the hybrid trp-lac promoter (Amannet al., Gene (1983) 25: 167; de Boer et al., Proc. Natl. Acad. Sci. USA(1983) 80: 21, and the bacteriophage T7 promoter (Studier et al., J.Mol. Biol. (1986); Tabor et al., (1985). These promoters and their useare discussed in Sambrook et al., supra. A presently preferred regulablepromoter is the dual tac-gal promoter, which is described inPCT/US97/20528 (Int'l. Publ. No. WO 9820111).

[0145] For expression of glycosyltransferase polypeptides in prokaryoticcells other than E. coli, a promoter that functions in the particularprokaryotic species is required. Such promoters can be obtained fromgenes that have been cloned from the species, or heterologous promoterscan be used. For example, a hybrid trp-lac promoter functions inBacillus in addition to E. coli. Promoters suitable for use ineukaryotic host cells are well known to those of skill in the art.

[0146] A ribosome binding site (RBS) is conveniently included in theexpression cassettes of the invention that are intended for use inprokaryotic host cells. An RBS in E. coli, for example, consists of anucleotide sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon (Shine and Dalgarno, Nature (1975) 254:34; Steitz, In Biological regulation and development: Gene expression(ed. R. F. Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, NY).

[0147] Translational coupling can be used to enhance expression. Thestrategy uses a short upstream open reading frame derived from a highlyexpressed gene native to the translational system, which is placeddownstream of the promoter, and a ribosome binding site followed after afew amino acid codons by a termination codon. Just prior to thetermination codon is a second ribosome binding site, and following thetermination codon is a start codon for the initiation of translation.The system dissolves secondary structure in the RNA, allowing for theefficient initiation of translation. See Squires et. al. (1988) J. Biol.Chem. 263: 16297-16302.

[0148] The glycosyltransferase polypeptides of the invention can beexpressed intracellularly, or can be secreted from the cell.Intracellular expression often results in high yields. If necessary, theamount of soluble, active glycosyltransferase polypeptides can beincreased by performing refolding procedures (see, e.g., Sambrook etal., supra.; Marston et al., Bio/Technology (1984) 2: 800; Schoner etal., Bio/Technology (1985) 3: 151). In embodiments in which theglycosyltransferase polypeptides are secreted from the cell, either intothe periplasm or into the extracellular medium, the polynucleotidesequence that encodes the glycosyltransferase is linked to apolynucleotide sequence that encodes a cleavable signal peptidesequence. The signal sequence directs translocation of theglycosyltransferase polypeptide through the cell membrane. An example ofa suitable vector for use in E. coli that contains a promoter-signalsequence unit is pTA1529, which has the E. coli phoA promoter and signalsequence (see, e.g., Sambrook et al., supra.; Oka et al., Proc. Natl.Acad. Sci. USA (1985) 82: 7212; Talmadge et al., Proc. Natl. Acad. Sci.USA (1980) 77: 3988; Takahara et al., J. Biol. Chem. (1985) 260: 2670).

[0149] The glycosyltransferase polypeptides of the invention can also beproduced as fusion proteins. This approach often results in high yields,because normal prokaryotic control sequences direct transcription andtranslation. In E. coli, lacZ fusions are often used to expressheterologous proteins. Suitable vectors are readily available, such asthe pUR, pEX, and pMR100 series (see, e.g., Sambrook et al., supra.).For certain applications, it may be desirable to cleave thenon-glycosyltransferase amino acids from the fusion protein afterpurification. This can be accomplished by any of several methods knownin the art, including cleavage by cyanogen bromide, a protease, or byFactor X_(a) (see, e.g., Sambrook et al., supra.; Itakura et al.,Science (1977) 198: 1056; Goeddel et al., Proc. Natl. Acad. Sci. USA(1979) 76: 106; Nagai et al., Nature (1984) 309: 810; Sung et al., Proc.Natl. Acad. Sci. USA (1986) 83: 561). Cleavage sites can be engineeredinto the gene for the fusion protein at the desired point of cleavage.

[0150] A suitable system for obtaining recombinant proteins from E. coliwhich maintains the integrity of their N-termini has been described byMiller et al. Biotechnology 7:698-704 (1989). In this system, the geneof interest is produced as a C-terminal fusion to the first 76 residuesof the yeast ubiquitin gene containing a peptidase cleavage site.Cleavage at the junction of the two moieties results in production of aprotein having an intact authentic N-terminal residue.

[0151] Glycosyltransferases of the invention can be expressed in avariety of host cells, including E. coli, other bacterial hosts, yeast,and various higher eukaryotic cells such as the COS, CHO and HeLa cellslines and myeloma-cell lines. Examples of useful bacteria include, butare not limited to, Escherichia, Enterobacter, Azotobacter, Erwinia,Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia,Shigella, Rhizobia, Vitreoscilla, and Paracoccus. The recombinantglycosyltransferase-encoding nucleic acid is operably linked toappropriate expression control sequences for each host. For E. coli thisincludes a promoter such as the T7, trp, or lambda promoters, a ribosomebinding site and preferably a transcription termination signal. Foreukaryotic cells, the control sequences will include a promoter andpreferably an enhancer derived from immunoglobulin genes, SV40,cytomegalovirus, etc., and a polyadenylation sequence, and may includesplice donor and acceptor sequences.

[0152] The expression vectors of the invention can be transferred intothe chosen host cell by well-known methods such as calcium chloridetransformation for E. coli and calcium phosphate treatment orelectroporation for mammalian cells. Cells transformed by the plasmidscan be selected by resistance to antibiotics conferred by genescontained on the plasmids, such as the amp, got, neo and hyg genes.

[0153] Once expressed, the recombinant glycosyltransferase polypeptidescan be purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y. (1990)). Substantially pure compositions of at least about 90to 95% homogeneity are preferred, and 98 to 99% or more homogeneity aremost preferred. Once purified, partially or to homogeneity as desired,the polypeptides may then be used (e.g., as immunogens for antibodyproduction). The glycosyltransferases can also be used in an unpurifiedor semi-purified state. For example, a host cell that expresses theglycosyltransferase can be used directly in a glycosyltransferasereaction, either with or without processing such as permeabilization orother cellular disruption.

[0154] One of skill would recognize that modifications can be made tothe glycosyltransferase proteins without diminishing their biologicalactivity. Some modifications may be made to facilitate the cloning,expression, or incorporation of the targeting molecule into a fusionprotein. Such modifications are well known to those of skill in the artand include, for example, a methionine added at the amino terminus toprovide an initiation site, or additional amino acids (e.g., poly His)placed on either terminus to create conveniently located restrictionsites or termination codons or purification sequences.

[0155] D. Methods and Reaction Mixtures for Synthesis ofOligosaccharides

[0156] The invention provides reaction mixtures and methods in which theglycosyltransferases of the invention are used to prepare desiredoligosaccharides (which are composed of two or more saccharides). Theglycosyltransferase reactions of the invention take place in a reactionmedium comprising at least one glycosyltransferase, a donor substrate,an acceptor sugar and typically a soluble divalent metal cation. Themethods rely on the use of the glycosyltransferase to catalyze theaddition of a saccharide to a substrate (also referred to as an“acceptor”) saccharide. A number of methods of usingglycosyltransferases to synthesize desired oligosaccharide structuresare known. Exemplary methods are described, for instance, WO 96/32491,Ito et al. (1993) Pure Appl. Chem. 65:753, and U.S. Pat. Nos. 5,352,670,5,374,541, and 5,545,553.

[0157] For example, the invention provides methods for adding sialicacid in an α2,3 linkage to a galactose residue, by contacting a reactionmixture comprising an activated sialic acid (e.g., CMP-NeuAc, CMP-NeuGc,and the like) to an acceptor moiety that includes a terminal galactoseresidue in the presence of a bifunctional sialyltransferase of theinvention. In presently preferred embodiments, the methods also resultin the addition of a second sialic acid residue which is linked to thefirst sialic acid by an α2,8 linkage. The product of this method isSiaα2,8-Siaα2,3-Gal-. Examples of suitable acceptors include a terminalGal that is linked to GlcNAc or Glc by a β1,4 linkage, and a terminalGal that is β1,3-linked to either GlcNAc or GalNAc. The terminal residueto which the sialic acid is attached can itself be attached to, forexample, H, a saccharide, oligosaccharide, or an aglycone group havingat least one carbohydrate atom. In some embodiments, the acceptorresidue is a portion of an oligosaccharide that is attached to aprotein, lipid, or proteoglycan, for example.

[0158] In some embodiments, the invention provides reaction mixtures andmethods for synthesis of gangliosides, lysogangliosides, gangliosidemimics, lysoganglioside mimics, or the carbohydrate portions of thesemolecules. These methods and reaction mixtures-typically include as thegalactosylated acceptor moiety a compound having a formula selected fromthe group consisting of Gal4Glc-R¹ and Gal3GalNAc-R²; wherein R¹ isselected from the group consisting of ceramide or other glycolipid, R²is selected from the group consisting of Gal4GlcCer,(Neu5Ac3)Gal4GlcCer, and (Neu5Ac8Neu5c3)Gal4GlcCer. For example, forganglioside synthesis the galactosylated acceptor can be selected fromthe group consisting of Gal4GlcCer, Gal3GalNAc4(Neu5Ac3)Gal4GlcCer, andGal3GalNAc4(Neu5Ac8Neu5c3) Gal4GlcCer.

[0159] The methods and reaction mixtures of the invention are useful forproducing any of a large number of gangliosides, lysogangliosides, andrelated structures. Many gangliosides of interest are described inOettgen, H. F., ed., Gangliosides and Cancer, VCH, Germany, 1989, pp.10-15, and references cited therein. Gangliosides of particular interestinclude, for example, those found in the brain as well as other sourceswhich are listed in Table 1. TABLE 1 Ganglioside Formulas andAbbreviations Structure Abbreviation Neu5Ac3Gal4GlcCer GM3GalNAc4(Neu5Ac3)Gal4GlcCer GM2 Gal3GalNAc4(Neu5Ac3)Gal4GlcCer GM1aNeu5Ac3Gal3GalNAc4Gal4GlcCer GM1b Neu5Ac8Neu5Ac3Gal4GlcCer GD3GalNAc4(Neu5Ac8Neu5Ac3)Gal4GlcCer GD2Neu5Ac3Gal3GalNAc4(Neu5Ac3)Gal4GlcCer GD1aNeu5Ac3Gal3(Neu5Ac6)GalNAc4Gal4GlcCer GD1αGal3GalNAc4(Neu5Ac8Neu5Ac3)Gal4GlcCer GD1bNeu5Ac8Neu5Ac3Gal3GalNAc4(Neu5Ac3)Gal4GlcCer GT1aNeu5Ac3Gal3GalNAc4(Neu5Ac8Neu5Ac3)Gal4GlcCer GT1bGal3GalNAc4(Neu5Ac8Neu5Ac8Neu5Ac3)Gal4GlcCer GT1cNeu5Ac8Neu5Ac3Gal3GalNAc4(Neu5Ac8Neu5c3)Gal4GlcCer GQ1b

[0160] The bifunctional sialyltransferases of the invention areparticularly useful for synthesizing the gangliosides GD1a, GD1b, GT1a,GT1b, GT1c, and GQ1b, or the carbohydrate portions of thesegangliosides, for example. The structures for these gangliosides, whichare shown in Table 1, requires both an α2,3- and anα2,8-sialyltransferase activity. An advantage provided by the methodsand reaction mixtures of the invention is that both activities arepresent in a single polypeptide.

[0161] The glycosyltransferases of the invention can be used incombination with additional glycosyltransferases and other enzymes. Forexample, one can use a combination of sialyltransferase andgalactosyltransferases. In some embodiments of the invention, thegalactosylated acceptor that is utilized by the bifunctionalsialyltransferase is formed by contacting a suitable acceptor withUDP-Gal and a galactosyltransferase. The galactosyltransferasepolypeptide, which can be one that is described herein, transfers theGal residue from the UDP-Gal to the acceptor.

[0162] Similarly, one can use the β1,4-GalNAc transferases of theinvention to synthesize an acceptor for the galactosyltransferase. Forexample, the acceptor saccharide for the galactosyltransferase canformed by contacting an acceptor for a GalNAc transferase withUDP-GalNAc and a GalNAc transferase polypeptide, wherein the GalNActransferase polypeptide transfers the GalNAc residue from the UDP-GalNActo the acceptor for the GalNAc transferase.

[0163] In this group of embodiments, the enzymes and substrates can becombined in an initial reaction mixture, or the enzymes and reagents fora second glycosyltransferase cycle can be added to the reaction mediumonce the first glycosyltransferase cycle has neared completion. Byconducting two glycosyltransferase cycles in sequence in a singlevessel, overall yields are improved over procedures in which anintermediate species is isolated. Moreover, cleanup and disposal ofextra solvents and by-products is reduced.

[0164] The products produced by the above processes can be used withoutpurification. However, it is usually preferred to recover the product.Standard, well known techniques for recovery of glycosylated saccharidessuch as thin or thick layer chromatography, or ion exchangechromatography. It is preferred to use membrane filtration, morepreferably utilizing a reverse osmotic membrane, or one or more columnchromatographic techniques for the recovery.

[0165] E. Uses of Glycoconjugates Produced Using Glycosyltransferasesand Methods of the Invention

[0166] The oligosaccharide compounds that are made using theglycosyltransferases and methods of the invention can be used in avariety of applications, e.g., as antigens, diagnostic reagents, or astherapeutics. Thus, the present invention also provides pharmaceuticalcompositions which can be used in treating a variety of conditions. Thepharmaceutical compositions are comprised of oligosaccharides madeaccording to the methods described above.

[0167] Pharmaceutical compositions of the invention are suitable for usein a variety of drug delivery systems. Suitable formulations for use inthe present invention are found in Remington 's Pharmaceutical Sciences,Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a briefreview of methods for drug delivery, see, Langer, Science 249:1527-1533(1990).

[0168] The pharmaceutical compositions are intended for parenteral,intranasal, topical, oral or local administration, such as by aerosol ortransdermally, for prophylactic and/or therapeutic treatment. Commonly,the pharmaceutical compositions are administered parenterally, e.g.,intravenously. Thus, the invention provides compositions for parenteraladministration which comprise the compound dissolved or suspended in anacceptable carrier, preferably an aqueous carrier, e.g., water, bufferedwater, saline, PBS and the like. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents and thelike.

[0169] These compositions may be sterilized by conventionalsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile aqueous carrierprior to administration. The pH of the preparations typically will bebetween 3 and 11, more preferably from 5 to 9 and most preferably from 7and 8.

[0170] In some embodiments the oligosaccharides of the invention can beincorporated into liposomes formed from standard vesicle-forming lipids.A variety of methods are available for preparing liposomes, as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S.Pat. Nos. 4,235,871, 4,501,728 and 4,837,028. The targeting of liposomesusing a variety of targeting agents (e.g., the sialyl galactosides ofthe invention) is well known in the art (see, e.g., U.S. Pat. Nos.4,957,773 and 4,603,044).

[0171] The compositions containing the oligosaccharides can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a patientalready suffering from a disease, as described above, in an amountsufficient to cure or at least partially arrest the symptoms of thedisease and its complications. An amount adequate to accomplish this isdefined as a “therapeutically effective dose.” Amounts effective forthis use will depend on the severity of the disease and the weight andgeneral state of the patient, but generally range from about 0.5 mg toabout 40 g of oligosaccharide per day for a 70 kg patient, with dosagesof from about 5 mg to about 20 g of the compounds per day being morecommonly used.

[0172] Single or multiple administrations of the compositions can becarried out with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the oligosaccharides of this invention sufficient toeffectively treat the patient.

[0173] The oligosaccharides may also find use as diagnostic reagents.For example, labeled compounds can be used to locate areas ofinflammation or tumor metastasis in a patient suspected of having aninflammation. For this use, the compounds can be labeled withappropriate radioisotopes, for example, ¹²⁵I, ¹⁴C, or tritium.

[0174] The oligosaccharide of the invention can be used as an immunogenfor the production of monoclonal or polyclonal antibodies specificallyreactive with the compounds of the invention. The multitude oftechniques available to those skilled in the art for production andmanipulation of various immunoglobulin molecules can be used in thepresent invention. Antibodies may be produced by a variety of means wellknown to those of skill in the art.

[0175] The production of non-human monoclonal antibodies, e.g., murine,lagomorpha, equine, etc., is well known and may be accomplished by, forexample, immunizing the animal with a preparation containing theoligosaccharide of the invention. Antibody-producing cells obtained fromthe immunized animals are immortalized and screened, or screened firstfor the production of the desired antibody and then immortalized. For adiscussion of general procedures of monoclonal antibody production, see,Harlow and Lane, Antibodies, A Laboratory Manual Cold Spring HarborPublications, N.Y. (1988).

EXAMPLE

[0176] The following example is offered to illustrate, but not to limitthe present invention.

[0177] This Example describes the use of two strategies for the cloningof four genes responsible for the biosynthesis of the GT1 a gangliosidemimic in the LOS of a bacterial pathogen, Campylobacter jejuni OH4384,which has been associated with Guillain-Barrésyndrome (Aspinall et al.(1994) Infect. Immun. 62: 2122-2125). Aspinal et al. ((1994)Biochemistry 33: 241-249) showed that this strain has an outer core LPSthat mimics the tri-sialylated ganglioside GT1a. We first cloned a geneencoding an α-2,3-sialyltransferase (cst-I) using an activity screeningstrategy. We then used raw nucleotide sequence information from therecently completed sequence of C. jejuni NCTC 11168 to amplify a regioninvolved in LOS biosynthesis from C. jejuni OH4384. Using primers thatare located in the heptosyl-transferases I and II, the 11.47 kb LOSbiosynthesis locus from C. jejuni OH4384 was amplified. Sequencingrevealed that the locus encodes 13 partial or complete open readingframes (ORFs), while the corresponding locus in C. jejuni NCTC 11168spans 13.49 kb and contains 15 ORFs, indicating a different organizationbetween these two strains.

[0178] Potential glycosyltransferase genes were cloned individually,expressed in Escherichia coli and assayed using synthetic fluorescentoligosaccharides as acceptors. We identified genes that encode aβ-1,4-N-acetylgalactosaminyl-transferase (cgtA), aβ-1,3-galactosyltransferase (cgtB) and a bifunctional sialyltransferase(cst-II) which transfers sialic acid to O-3 of galactose and to O-8 of asialic acid that is linked α-2,3-to a galactose. The linkage specificityof each identified glycosyltransferase was confirmed by NMR analysis at600 MHz on nanomole amounts of model compounds synthesized in vitro.Using a gradient inverse broadband nano-NMR probe, sequence informationcould be obtained by detection of ³J(C, H) correlations across theglycosidic bond. The role of cgtA and cst-II in the synthesis of theGT1a mimic in C. jejuni QH4384 were confirmed by comparing theirsequence and activity with corresponding homologues in two related C.jejuni strains that express shorter ganglioside mimics in their LOS.Thus, these three enzymes can be used to synthesize a GT1a mimicstarting from lactose.

[0179] The abbreviations used are: CE, capillary electrophoresis;CMP-Neu5Ac, cytidine monophosphate-N-acetylneuraminic acid; COSY,correlated spectroscopy; FCHASE, 6-(5-fluorescein-carboxamido)-hexanoicacid succimidyl ester; GBS, Guillain-Barré syndrome; HMBC, heteronuclearmultiple bond coherence; HSQC, heteronuclear single quantum coherence;LIF, laser induced fluorescence; LOS, lipooligosaccharide; LPS,lipopolysaccharide; NOE, nuclear Overhauser effect; NOESY, NOEspectroscopy; TOCSY, total correlation spectroscopy.

[0180] Experimental Procedures

[0181] Bacterial Strains

[0182] The following C. jejuni strains were used in this study:serostain O:19 (ATCC #43446); serotype O:19 (strains OH4382 and OH4384were obtained from the Laboratory Centre for Disease Control (HealthCanada, Winnipeg, Manitoba)); and serotype O:2 (NCTC #11168).Escherichia coli DH5α was used for the HindE library while E. coli AD202(CGSG #7297) was used to express the different clonedglycosyltransferases.

[0183] Basic Recombinant DNA Methods.

[0184] Genomic DNA isolation from the C. jejuni strains was performedusing Qiagen Genomic-tip 500/G (Qiagen Inc., Valencia, Calif.) asdescribed previously (Gilbert et al. (1996) J. Biol. Chem. 271:28271-28276). Plasmid DNA isolation, restriction enzyme digestions,purification of DNA fragments for cloning, ligations and transformationswere performed as recommended by the enzyme supplier, or themanufacturer of the kit used for the particular procedure. Long PCRreactions (>3 kb) were performed using the Expand™ long template PCRsystem as described by the manufacturer (Boehringer Mannheim, Montreal).PCR reactions to amplify specific ORFs were performed using the Pwo DNApolymerase as described by the manufacturer (Boehringer Mannheim,Montreal). Restriction and DNA modification enzymes were purchased fromNew England Biolabs Ltd. (Mississauga, ON). DNA sequencing was performedusing an Applied Biosystems (Montreal) model 370A automated DNAsequencer and the manufacturer's cycle sequencing kit.

[0185] Activity Screening for Sialyltransferase from C. jejuni

[0186] The genomic library was prepared using a partial HindIII digestof the chromosomal DNA of C. jejuni OH4384. The partial digest waspurified on a QIAquick column (QIAGEN Inc.) and ligated with HindIIIdigested pBluescript SK-. E. coli DH5α was electroporated with theligation mixture and the cells were plated on LB medium with 150 μg/mLampicillin, 0.05 mM IPTG and 100 μg/mL X-Gal(5-Bromo4-chloro-indolyl-β-D-galactopyranoside). White colonies werepicked in pools of 100 and were resuspended in 1 mL of medium with 15%glycerol. Twenty μL of each pool were used to inoculate 1.5 mL of LBmedium supplemented with 150 μg/mL ampicillin. After 2 h of growth at37° C., IPTG was added to 1 mM and the cultures were grown for another4.5 h. The cells were recovered by centrifugation, resuspended in 0.5 mLof 50 mM Mops (pH 7, 10 mM MgCl₂) and sonicated for 1 min. The extractswere assayed for sialyltransferase activity as described below exceptthat the incubation time and temperature were 18 h and 32° C.,respectively. The positive pools were plated for single colonies, and200 colonies were picked and tested for activity in pools of 10. Finallythe colonies of the positive pools were tested individually which led tothe isolation of a two positive clones, pCJH9 (5.3 kb insert) andpCJH101 (3.9 kb insert). Using several sub-cloned fragments andcustom-made primers, the inserts of the two clones were completelysequenced on both strands. The clones with individual HindIII fragmentswere also tested for sialyltransferase activity and the insert of theonly positive one (a 1.1 kb HindIII fragment cloned in pBluescript SK-)was transferred to pUC118 using KpnI and PstI sites in order to obtainthe insert in the opposite orientation with respect to the placpromoter.

[0187] Cloning and Sequencing of the LPS Biosynthesis Locus.

[0188] The primers used to amplify the LPS biosynthesis locus of C.jejuni OH4384 were based on preliminary sequences available from thewebsite (URL: http://www.sanger.ac.uk/Projects/C_jejuni/) of the C.jejuni sequencing group (Sanger Centre, UK) who sequenced the completegenome of the strain NCTC11168. The primers CJ-42 and CJ-43 (all primerssequences are described in Table 2) were used to amplify an 11.47 kblocus using the Expand™ long template PCR system. The PCR product waspurified on a S-300 spin column (Pharmacia Biotech) and completelysequence on both strands using a combination of primer walking andsub-cloning of HindIII fragments. Specific ORF's were amplified usingthe primers described in Table 2 and the Pwo DNA polymerase. The PCRproducts were digested using the appropriate restriction enzymes (seeTable 2) and were cloned in pCWori+. TABLE 2 Primers used forAmplification of Open Reading Frames Primers used to amplify the LPScore biosynthesis locus CJ42: Primer in heptosylTase-II(Error! Reference source not found.) 5′ GC CAT TAC CGT ATC GCC TAA CCAGG 3′  25 mer CJ43: Primer in heptosylTase-I (Error! Reference sourcenot found.) 5′ AAA GAA TAC GAA TTT GCT AAA GAG G 3′  25 mer Primers usedto amplify and clone ORF 5a: CJ-106 (3′ primer, 41 mer)(Error! Referencesource not found.):           SalI 5′ CCT AGG TCG ACT TAA AAC AAT GTTAAG AAT ATT TTT TTT AG 3′ CJ-157 (5′ primer, 37 mer)(Error! Referencesource not found.):                   NdeI 5′ CTT AGG AGG TCA TAT GCTATT TCA ATC ATA CTT TGT G 3′ Primers used to amplify and clone ORF 6a:CJ-105 (3′ primer, 37 mer)(Error! Reference source not found.):          SalI 5′ CCT AGG TCG ACC TCT AAA AAA AAT ATT CTT AAC ATT G 3′CJ-133 (5′ primer, 39 mer)(Error! Reference source not found.):              NdeI 5′ CTTAGGAGGTCATATGTTTAAAATTTCAATCATCTTACC 3′ Primersused to amplify and clone ORF 7a: CJ-131 (5′ primer, 41mer)(Error! Reference source not found.):               NdeI5′ CTTAGGAGGTCATATGAAAAAAGTTATTATTGCTGGAAATG 3′ CJ-132 (3′ primer, 41mer)(Error! Reference source not found.):          SalI5′ CCTAGGTCGACTTATTTTCCTTTGAAATAATGCTTTATATC 3′

[0189] Expression in E. coli and glycosyltransferase assays.

[0190] The various constructs were transferred to E. coli AD202 and weretested for the expression of glycosyltransferase activities following a4 h induction with 1 mM IPTG. Extracts were made by sonication and theenzymatic reactions were performed overnight at 32° C. FCHASE-labeledoligosaccharides were prepared as described previously (Wakarchuk et al.(1996) J. Biol. Chem. 271: 19166-19173). Protein concentration wasdetermined using the bicinchoninic acid protein assay kit (Pierce,Rockford, Ill.). For all of the enzymatic assays one unit of activitywas defined as the amount of enzyme that generated one μmol of productper minute.

[0191] The screening assay for α-2,3-sialyltransferase activity in poolsof clones contained 1 mM Lac-FCHASE, 0.2 mM CMP-Neu5Ac, 50 mM Mops pH 7,10 mM MnCl₂ and 10 mM MgCl₂ in a final volume of 10 μL. The varioussubcloned ORFs were tested for the expression of glycosyltransferaseactivities following a 4 h induction of the cultures with 1 mM IPTG.Extracts were made by sonication and the enzymatic reactions wereperformed overnight at 32° C.

[0192] The β-1,3-galactosyltransferase was assayed using 0.2 mMGM2-FCHASE, 1 mM UDP-Gal, 50 mM Mes pH 6, 10 mM MnCl₂ and 1 mM DTT. Theβ-1,4-GalNAc transferase was assayed using 0.5 mM GM3-FCHASE, 1 mMUDP-GalNAc, 50 mM Hepes pH 7 and 10 mM MnCl₂. Theα-2,3-sialyltransferase was assayed using 0.5 mM Lac-FCHASE, 0.2 mMCMP-Neu5Ac, 50 mM Hepes pH 7 and 10 mM MgCl₂. Theα-2,8-sialyltransferase was assayed using 0.5 mM GM3-FCHASE, 0.2 mMCMP-Neu5Ac, 50 mM Hepes pH 7 and 10 mM MnCl₂.

[0193] The reaction mixes were diluted appropriately with 10 mM NaOH andanalyzed by capillary electrophoresis performed using the separation anddetection conditions as described previously (Gilbert et al. (1996) J.Biol. Chem. 271, 28271-28276). The peaks from the electropherograms wereanalyzed using manual peak integration with the P/ACE Station software.For rapid detection of enzyme activity, samples from the transferasereaction mixtures were examined by thin layer chromatography onsilica-60 TLC plates (E. Merck) as described previously (Id.).

[0194] NMR Spectroscopy

[0195] NMR experiments were performed on a Varian INOVA 600 NMRspectrometer. Most experiments were done using a 5 mm Z gradient tripleresonance probe. NMR samples were prepared from 0.3-0.5 mg (200-500nanomole) of FCHASE-glycoside. The compounds were dissolved in H₂O andthe pH was adjusted to 7.0 with dilute NaOH. After freeze drying thesamples were dissolved in 600 μL D₂O. All NMR experiments were performedas previously described (Pavliak et al. (1993) J. Biol. Chem. 268:14146-14152; Brisson et al. (1997) Biochemistry 36: 3278-3292) usingstandard techniques such as COSY, TOCSY, NOESY, 1D-NOESY, 1D-TOCSY andHSQC. For the proton chemical shift reference, the methyl resonance ofinternal acetone was set at 2.225 ppm (¹H). For the ¹³C chemical shiftreference, the methyl resonance of internal acetone was set at 31.07 ppmrelative to external dioxane at 67.40 ppm. Homonuclear experiments wereon the order of 5-8 hours each. The 1D NOESY experiments for GD3-FCHASE,[0.3 mM], with 8000 scans and a mixing time of 800 ms was done for aduration of 8.5 h each and processed with a line broadening factor of2-5 Hz. For the 1D NOESY of the resonances at 4.16 ppm, 3000 scans wereused. The following parameters were used to acquire the HSQC spectrum:relaxation delay of 1.0 s, spectral widths in F₂ and F₁ of 6000 and24147 Hz, respectively, acquisition times in t₂ of 171 ms. For the t₁dimension, 128 complex points were acquired using 256 scans perincrement. The sign discrimination in F₁ was achieved by the Statesmethod. The total acquisition time was 20 hours. For GM2-FCHASE, due tobroad lines, the number of scans per increment was increased so that theHSQC was performed for 64 hours. The phase-sensitive spectrum wasobtained after zero filling to 2048×2048 points. Unshifted gaussianwindow functions were applied in both dimensions. The HSQC spectra wereplotted at a resolution of 23 Hz/point in the ¹³C dimension and 8Hz/point in the proton dimension. For the observation of the multipletsplittings, the ¹H dimension was reprocessed at a resolution of 2Hz/point using forward linear prediction and a π/4-shifted squaredsinebell function. All the NMR data was acquired using Varian's standardsequences provided with the VNMR 5.1 or VNMR 6.1 software. The sameprogram was used for processing.

[0196] A gradient inverse broadband nano-NMR probe (Varian) was used toperform the gradient HMBC (Bax and Summers (1986) J. Am. Chem. Soc. 108,2093-2094; Parella et al. (1995) J. Mag. Reson. A 112, 241-245)experiment for the GD3-FCHASE sample. The nano-NMR probe which is ahigh-resolution magic angle spinning probe produces high resolutionspectra of liquid samples dissolved in only 40 μL (Manzi et al. (1995)J. Biol. Chem. 270, 9154-9163). The GD3-FCHASE sample (mass=1486.33 Da)was prepared by lyophilizing the original 0.6 mL sample (200 nanomoles)and dissolving it in 40 μL of D₂O for a final concentration of 5 mM. Thefinal pH of the sample could not be measured.

[0197] The gradient HMBC experiment was done at a spin rate of 2990 Hz,400 increments of 1024 complex points, 128 scans per increment,acquisition time of 0.21 s, ¹J(C, H)=140 Hz and ^(n)J(C, H)=8 Hz, for aduration of 18.5 h.

[0198] Mass Spectrometry

[0199] All mass measurements were obtained using a Perkin-ElmerBiosystems (Fragmingham, Mass.) Elite-STR MALDI-TOF instrument.Approximately two μg of each oligosaccharide was mixed with a matrixcontaining a saturated solution of dihydroxybenzoic acid. Positive andnegative mass spectra were acquired using the reflector mode.

[0200] Results

[0201] Detection of glycosyltransferase Activities in C. jejuni Strains

[0202] Before the cloning of the glycosyltransferase genes, we examinedC. jejuni OH4384 and NCTC 11168 cells for various enzymatic activities.When an enzyme activity was detected, the assay conditions wereoptimized (described in the Experimental Procedures) to ensure maximalactivity. The capillary electrophoresis assay we employed was extremelysensitive and allowed detection of enzyme activity in the μU/ml range(Gilbert et al. (1996) J. Biol. Chem. 271: 28271-28276). We examinedboth the sequenced strain NCTC 11168 and the GBS-associated strainOH4384 for the enzymes required for the GT1a ganglioside mimicsynthesis. As predicted, strain OH4384 possessed the enzyme activitiesrequired for the synthesis of this structure:β-1,4-N-acetylgalactosaminyltransferase, β-1,3-galactosyltransferase,β-2,3-sialyltransferase and α-2,8-sialyltransferase. The genome of thestrain, NCTC 11168 lacked the β-1,3-galactosyltransferase and theα-2,8-sialyltransferase activities.

[0203] Cloning of an α-2,3-sialyltransferase (cst-I) Using an ActivityScreening Strategy

[0204] A plasmid library made from an unfractionated partial HindIIIdigestion of chromosomal DNA from C. jejuni OH4384 yielded 2,600 whitecolonies which were picked to form pools of 100. We used a “divide andconquer” screening protocol from which two positive clones were obtainedand designated pCJH9 (5.3 kb insert, 3 HindIII sites) and pCJH101 (3.9kb insert, 4 HindIII sites). Open reading frame (ORF) analysis and PCRreactions with C. jejuni OH4384 chromosomal DNA indicated that pCJH9contained inserts that were not contiguous in the chromosomal DNA. Thesequence downstream of nucleotide #1440 in pCJH9 was not further studiedwhile the first 1439 nucleotides were found to be completely containedwithin the sequence of pCJH101. The ORF analysis and PCR reactions withchromosomal DNA indicated that all of the pCJH101 HindIII fragments werecontiguous in C. jejuni OH4384 chromosomal DNA.

[0205] Four ORFs, two partial and two complete, were found in thesequence of pCJH101 (FIG. 2). The first 812 nucleotides encode apolypeptide that is 69% identical with the last 265 a.a. residues of thepeptide chain release factor RF-2 (prfB gene, GenBank #AE000537) fromHelicobacter pylori. The last base of the TAA stop codon of the chainrelease factor is also the first base of the ATG start codon of an openreading frame that spans nucleotides #812 to #2104 in pCJH101. This ORFwas designated cst-I (Campylobacter sialyltransferase I) and encodes a430 amino acid polypeptide that is homologous with a putative ORF fromHaemophilus influenzae (GenBank #U32720). The putative H. influenzae ORFencodes a 231 amino acid polypeptide that is 39% identical to the middleregion of the Cst I polypeptide (amino acid residues #80 to #330). Thesequence downstream of cst-I includes an ORF and a partial ORF thatencode polypeptides that are homologous (>60% identical) with the twosubunits, CysD and CysN, of the E. coli sulfate adenylyltransferase(GenBank #AE000358).

[0206] In order to confirm that the cst-I ORE encodes sialyltransferaseactivity, we sub-cloned it and over-expressed it in E. coli. Theexpressed enzyme was used to add sialic acid to Gal-β-1,4-Glc-β-FCHASE(Lac-FCHASE). This product (GM3-FCHASE) was analyzed by NMR to confirmthe Neu5Ac-α-2,3-Gal linkage specificity of Cst-I.

[0207] Sequencing of the LOS biosynthesis locus of C. jejuni OH4384

[0208] Analysis of the preliminary sequence data available at thewebsite of the C. jejuni NCTC 11168 sequencing group (Sanger Centre, UK(http://www.sanger.ac.ukProjects/C_jejuni/)) revealed that the twoheptosyltransferases involved in the synthesis of the inner core of theLPS were readily identifiable by sequence homology with other bacterialheptosyltransferases. The region between the two heptosyltransferasesspans 13.49 kb in NCTC 11168 and includes at least seven potentialglycosyltransferases based on BLAST searches in GenBank. Since nostructure is available for the LOS outer core of NCTC 11168, it wasimpossible to suggest functions for the putative glycosyltransferasegenes in that strain.

[0209] Based on conserved regions in the heptosyltransferases sequences,we designed primers (CJ-42 and CJ-43) to amplify the region betweenthem. We obtained a PCR product of 13.49 kb using chromosomal DNA fromC. jejuni NCTC 11168 and a PCR product of 11.47 kb using chromosomal DNAfrom C. jejuni OH4384. The size of the PCR product from strain NCTC11168 was consistent with the Sanger Centre data. The smaller size ofthe PCR product from strain OH4384 indicated heterogeneity between thestrains in the region between the two heptosyltransferase genes andsuggested that the genes for some of the glycosyltransferases specificto strain OH4384 could be present in that location. We sequenced the11.47 kb PCR product using a combination of primer walking andsub-cloning of HindIII fragments (GenBank #AF 130984). The G/C contentof the DNA was 27%, typical of DNA from Campylobacter. Analysis of thesequence revealed eleven complete ORFs in addition to the two partialORFs encoding the two heptosyltransferases (FIG. 2, Table 3). Whencomparing the deduced amino acid sequences, we found that the twostrains share six genes that are above 80% identical and four genes thatare between 52 and 68% identical (Table 3). Four genes are unique to C.jejuni NCTC 11168 while one gene is unique to C. jejuni OH4384 (FIG. 2).Two genes that are present as separate ORFs (ORF #5a and #10a) in C.jejuni OH4384 are found in an in-frame fusion ORF (#5b/10b) in C. jejuniNCTC 11168. TABLE 3 Location and description of the ORFs of the LOSbiosynthesis locus from C. jejuni OH4384 Homologue in StrainNCTC11168^(a) (% identity Homologues found in in the GenBank a.a. (%identity in the a.a ORF # Location sequence) sequence) Function^(b)  1a   1-357 ORF #1b rfaC (GB #AE000546) Heptosyltransferase I (98%) fromHelicobacter pylori (35%)  2a   350-1,234 ORF #2b waaM (GB Lipid Abiosynthesis (96%) #AE001463) from acyltransferase Helicobacter pylori(25%)  3a  1,234-2,487 ORF #3b lgtF (GB #U58765) Glycosyltransferase(90%) from Neisseria meningitidis (31%)  4a  2,786-3,952 ORF #4b cps14J(GB #X85787) Glycosyltransferase (80%) from Streptococcus pneumoniae(45% over first 100 a.a)  5a  4,025-5,065 N-terminus of ORF #HP0217 (GBβ-1,4-N-acetylgalac- ORF #5b/10b #AE000541) tosaminyltransferase (52%)from Helicobacter (cgtA) pylori (50%)  6a  5,057-5,959 ORF #6b cps23FU(GB β-1,3-Galactosyltransferase (complement) (60%) #AF030373) from(cgtB) Streptococcus pneumoniae (23%)  7a  6,048-6,920 ORF #7b ORF#HI0352 (GB Bi-functional α- (52%) #U32720) from 2,3/α2,8sialyltransferase Haemophilus (cst-II) influenzae (40%)  8a  6,924-7,961ORF #8b siaC (GB #U40740) Sialic acid synthase (80%) from Neisseriameningitidis (56%)  9a  8,021-9,076 ORF #9b siaA (GB #M95053) Sialicacid biosynthesis (80%) from Neisseria meningitidis (40%) 10a 9,076-9,738 C-terminus of neuA (GB #U54496) CMP-sialic acid ORF #5b/10bfrom synthetase (68%) Haemophilus ducreyi (39%) 11a  9,729-10,559 NoPutative ORF (GB Acetyltransferase homologue #AF010496) from Rhodobactercapsulatus (22%) 12a 10,557-11,366 ORF #12b ORF #HI0868 (GBGlycosyltransferase (complement) (90%) #U32768) from Haemophilusinfluenzae (23%) 13a 11,347-11,474 ORF #13b rfaF (GB #AE000625)Heptosyltransferase II (100%) from Helicobacter pylori (60%)

[0210] Identification of Outer Core Glycosyltransferases

[0211] Various constructs were made to express each of the potentialglycosyltransferase genes located between the two heptosyltransferasesfrom C. jejuni OH4384. The plasmid pCJL-09 contained the ORF #5a and aculture of this construct showed GalNAc transferase activity whenassayed using GM3-FCHASE as acceptor. The GalNAc transferase wasspecific for a sialylated acceptor since Lac-FCHASE was a poor substrate(less than 2% of the activity observed with GM3-FCHASE). The reactionproduct obtained from GM3-FCHASE had the correct mass as determined byMALDI-TOF mass spectrometry, and the identical elution time in the CEassay as the GM2-FCHASE standard. Considering the structure of the outercore LPS of C. jejuni OH4384, this GalNAc transferase (cgtA forCamplyobacter glycosyltransferase A), has a β-1,4-specificity to theterminal Gal residue of GM3-FCHASE. The linkage specificity of CgtA wasconfirmed by the NMR analysis of GM2-FCHASE (see text below, Table 4).The in vivo role of cgtA in the synthesis of a GM2 mimic is confirmed bythe natural knock-out mutant provided by C. jejuni OH4382 (FIG. 1). Uponsequencing of the cgtA homologue from C. jejuni OH4382 we found aframe-shift mutation (a stretch of seven A's instead of 8 A's after base#71) which would result in the expression of a truncated cgtA version(29 aa instead of 347 aa). The LOS outer core structure of C. jejuniOH4382 is consistent with the absence of β-1,4-GlaNAc transferase as theinner galactose residue is substituted with sialic acid only (Aspinallet al. (1994) Biochemistry 33, 241-249).

[0212] The plasmid pCJL-04 contained the ORF #6a and an IPTG-inducedculture of this construct showed galactosyltransferase activity usingGM2-FCHASE as an acceptor thereby producing GM1a-FCHASE. This productwas sensitive to β-1,3-galactosidase and was found to have the correctmass by MALDI-TOF mass spectrometry. Considering the structure of theLOS outer core of C. jejuni OH4384, we suggest that thisgalactosyltransferase (cgtB for Campylobacter glycosyltransferase B) hasβ-1,3-specificity to the terminal GalNAc residue of GM2-FCHASE. Thelinkage specificity of CgtA was confirmed by the NMR analysis ofGM1a-FCHASE (see text below, Table 4) which was synthesized by usingsequentially Cst-I, CgtA and CgtB.

[0213] The plasmid pCJL-03 included the ORF #7a and an IPTG-inducedculture showed sialyltransferase activity using both Lac-FCHASE andGM3-FCHASE as acceptors. This second sialyltransferase from OH4384 wasdesignated cst-II. Cst-II was shown to be bi-functional as it couldtransfer sialic acid α-2,3 to the terminal Gal of Lac-FCHASE and alsoα-2,8-to the terminal sialic acid of GM3-FCHASE. NMR analysis of areaction product formed with Lac-FCHASE confirmed the α-2,3-linkage ofthe first sialic acid on the Gal, and the α-2,8-linkage of the secondsialic acid (see text below, Table 4). TABLE 4 Proton NMR chemicalshifts^(a) for the fluorescent derivatives of the ganglioside mimicssynthesized using the cloned glycosyltransferases. Chemical Shift (ppm)Residue H Lac- GM3- GM2- GM1a- GD3- βGlc 1 4.57 4.70 4.73 4.76 4.76 a 23.23 3.32 3.27 3.30 3.38 3 3.47 3.54 3.56 3.58 3.57 4 3.37 3.48 3.393.43 3.56 5 3.30 3.44 3.44 3.46 3.50 6 3.73 3.81 3.80 3.81 3.85 6′ 3.223.38 3.26 3.35 3.50 βGal(1-4) 1 4.32 4.43 4.42 4.44 4.46 b 2 3.59 3.603.39 3.39 3.60 3 3.69 4.13 4.18 4.18 4.10 4 3.97 3.99 4.17 4.17 4.00 53.81 3.77 3.84 3.83 3.78 6 3.86 3.81 3.79 3.78 3.78 6′ 3.81 3.78 3.793.78 3.78 αNeu5Ac(2-3) 3_(ax) 1.81 1.97 1.96 1.78 c 3_(eq) 2.76 2.672.68 2.67 4 3.69 3.78 3.79 3.60 5 3.86 3.84 3.83 3.82 6 3.65 3.49 3.513.68 7 3.59 3.61 3.60 3.87 8 3.91 3.77 3.77 4.15 9 3.88 3.90 3.89 4.189′ 3.65 3.63 3.64 3.74 NAc 2.03 2.04 2.03 2.07 βGalNAc(1-4) 1 4.77 4.81d 2 3.94 4.07 3 3.70 3.82 4 3.93 4.18 5 3.74 3.75 6 3.86 3.84 6′ 3.863.84 NAc 2.04 2.04 βGal(1-3) 1 4.55 e 2 3.53 3 3.64 4 3.92 5 3.69 6 3.786′ 3.74 αNeu5Ac(2-8) 3_(ax) 1.75 f 3_(eq) 2.76 4 3.66 5 3.82 6 3.61 73.58 8 3.91 9 3.88 9′ 3.64 NAc 2.02

[0214] Comparison of the Sialyltransferases

[0215] The in vivo role of cst-II from C. jejuni OH4384 in the synthesisof a tri-sialylated GT1a ganglioside mimic is supported by comparisonwith the cst-II homologue from C. jejuni O:19 (serostrain) thatexpresses the di-sialylated GD1a ganglioside mimic. There are 24nucleotide differences that translate into 8 amino acid differencesbetween these two cst-II homologues (FIG. 3). When expressed in E. coli,the cst-II homologue from C. jejuni O:19 (serostrain) hasα-2,3-sialyltransferase activity but very low α-2,8-sialyltransferaseactivity (Table 5) which is consistent with the absence of terminalα-2,8-linked sialic acid in the LOS outer core (Aspinall et al. (1994)Biochemistry 33, 241-249) of C. jejuni O:19 (serostrain). The cst-IIhomologue from C. jejuni NCTC 11168 expressed much lowerα-2,3-sialyltransferase activity than the homologues from O:19(serostrain) or OH4384 and no detectable α-2,8-sialyltransferaseactivity. We could detect an IPTG-inducible band on a SDS-PAGE gel whencst-II from NCTC 11168 was expressed in E. coli (data not shown). TheCst-II protein from NCTC 11168 shares only 52% identity with thehomologues from O:19 (serostrain) or OH4384. We could not determinewhether the sequence differences could be responsible for the loweractivity expressed in E. coli.

[0216] Although cst-I mapped outside the LOS biosynthesis locus, it isobviously homologous to cst-II since its first 300 residues share 44%identity with Cst-II from either C. jejuni OH4384 or C. jejuni NCTC11168 (FIG. 3). The two Cst-II homologues share 52% identical residuesbetween themselves and are missing the C-terminal 130 amino acids ofCst-I. A truncated version of Cst-I which was missing 102 amino acids atthe C-terminus was found to be active (data not shown) which indicatesthat the C-terminal domain of Cst-I is not necessary forsialyltransferase activity. Although the 102 residues at the C-terminusare dispensable for in vitro enzymatic activity, they may interact withother cell components in vivo either for regulatory purposes or forproper cell localization. The low level of conservation between the C.jejuni sialyltransferases is very different from what was previouslyobserved for the α-2,3-sialyltransferases from N. meningitidis and N.gonorrhoeae, where the 1st transferases are more than 90% identical atthe protein level between the two species and between different isolatesof the same species (Gilbert et al., supra.). TABLE 5 Comparison of theactivity of the sialyltransferases from C. jejuni. The varioussialyltransferases were expressed in E. coli as fusion proteins with themaltose-binding protein in the vector pCWori+ (Wakarchuk et al. (1994)Protein. Sci. 3, 467-475). Sonicated extracts were assayed using 500 μMof either Lac-FCHASE or GM3-FCHASE. Sialyltransferase Activity(μU/mg)^(a) gene Lac-FCHASE GM3-FCHASE Ratio (%)^(b) cst-I (OH4384)3,744 2.2 0.1 cst-II (OH4384)   209 350.0 167.0 cst-II (O:19 serostrain)2,084 1.5 0.1 cst-II (NCTC 11168)    8 0 0.0

[0217] NMR Analysis on Nanomole Amounts of the Synthesized ModelCompounds.

[0218] In order to properly assess the linkage specificity of anidentified glycosyltransferase, its product was analyzed by NMRspectroscopy. In order to reduce the time needed for the purification ofthe enzymatic products, NMR analysis was conducted on nanomole amounts.All compounds are soluble and give sharp resonances with linewidths of afew Hz since the H-1 anomeric doublets (J_(1,2) =8 Hz) are wellresolved. The only exception is for GM2-FCHASE which has broad lines(˜10 Hz), probably due to aggregation. For the proton spectrum of the 5mM GD3-FCHASE solution in the nano-NMR probe, the linewidths of theanomeric signals were on the order of 4 Hz, due to the increasedconcentration. Also, additional peaks were observed, probably due todegradation of the sample with time. There were also some slightchemical shifts changes, probably due to a change in pH uponconcentrating the sample from 0.3 mM to 5 mM. Proton spectra wereacquired at various temperatures in order to avoid overlap of the HDOresonance with the anomeric resonances. As can be assessed from theproton spectra, all compounds were pure and impurities or degradationproducts that were present did not interfere with the NMR analysis whichwas performed as previously described (Pavliak et al. (1993) J. Biol.Chem. 268, 14146-14152; Brisson et al. (1997) Biochemistry 36,3278-3292).

[0219] For all of FCHASE glycosides, the ¹³C assignments of similarglycosides (Sabesan and Paulson (1986) J. Am. Chem. Soc. 108, 2068-2080;Michon et al. (1987) Biochemistry 26, 8399-8405; Sabesan et al. (1984)Can. J. Chem. 62, 1034-1045) were available. For the FCHASE glycosides,the ¹³C assignments were verified by first assigning the proton spectrumfrom standard homonuclear 2D experiments, COSY, TOCSY and NOESY, andthen verifying the ¹³C assignments from an HSQC experiment, whichdetects C—H correlations. The HSQC experiment does not detect quaternarycarbons like C-1 and C-2 of sialic acid, but the HMBC experiment does.Mainly for the Glc resonances, the proton chemical shifts obtained fromthe HSQC spectra differed from those obtained from homonuclearexperiments due to heating of the sample during ¹³C decoupling. From aseries of proton spectrum acquired at different temperatures, thechemical shifts of the Glc residue were found to be the most sensitiveto temperature. In all compounds, the H-1 and H-2 resonances of Glcchanged by 0.004 ppm/° C., the Gal(1-4) H-1 by 0.002 ppm/° C., and lessthan 0.001 ppm/° C. for the Neu5Ac H-3 and other anomeric resonances.For LAC-FCHASE, the Glc H-6 resonance changed by 0.008 ppm/° C.

[0220] The large temperature coefficient for the Glc resonances isattributed to ring current shifts induced by the linkage to theaminophenyl group of FCHASE. The temperature of the sample during theHSQC experiment was measured from the chemical shift of the Glc H-1 andH-2 resonances. For GM1a-FCHASE, the temperature changed from 12° C. to24° C. due to the presence of the Na+ counterion in the solution andNaOH used to adjust the pH. Other samples had less severe heating (<5°C.). In all cases, changes of proton chemical shifts with temperaturedid not cause any problems in the assignments of the resonances in theHSQC spectrum. In Table 4 and Table 6, all the chemical shifts are takenfrom the HSQC spectra.

[0221] The linkage site on the aglycon was determined mainly from acomparison of the ¹³C chemical shifts of the enzymatic product withthose of the precursor to determine glycosidation shifts as donepreviously for ten sialyloligosaccharides (Salloway et al. (1996)Infect. Immun. 64, 2945-2949). Here, instead of comparing ¹³C spectra,HSQC spectra are compared, since one hundred times more material wouldbe needed to obtain a ¹³C spectrum. When the ¹³C chemical shifts fromHSQC spectra of the precursor compound are compared to those of theenzymatic product, the main downfield shift always occurs at the linkagesite while other chemical shifts of the precursor do not changesubstantially. Proton chemical shift differences are much moresusceptible to long-range conformational effects, sample preparation,and temperature. The identity of the new sugar added can quickly beidentified from a comparison of its ¹³C chemical shifts with those ofmonosaccharides or any terminal residue, since only the anomericchemical shift of the glycon changes substantially upon glycosidation(Sabesan and Paulson, supra.).

[0222] Vicinal proton spin-spin coupling (J_(HH)) obtained from 1D TOCSYor 1D NOESY experiments also are used to determine the identity of thesugar. NOE experiments are done to sequence the sugars by theobservation of NOEs between the anomeric glycon protons (H-3s for sialicacid) and the aglycon proton resonances. The largest NOE is usually onthe linkage proton but other NOEs can also occur on aglycon protonresonances that are next to the linkage site. Although at 600 MHz, theNOEs of many tetra- and pentasaccharides are positive or very small, allthese compounds gave good negative NOEs with a mixing time of 800 ms,probably due to the presence of the large FCHASE moiety.

[0223] For the synthetic Lac-FCHASE, the ¹³C assignments for the lactosemoiety of Lac-FCHASE were confirmed by the 2D methods outlined above.All the proton resonances of the Glc unit were assigned from a 1D-TOCSYexperiment on the H-1 resonance of Glc with a mixing time of 180 ms. AID-TOCSY experiment for Gal H-1 was used to assign the H-1 to H-4resonances of the Gal unit. The remaining H-5 and H-6s of the Gal unitwere then assigned from the HSQC experiment. Vicinal spin-spin couplingvalues (J_(HH)) for the sugar units were in accord with previous data(Michon et al., supra.). The chemical shifts for the FCHASE moiety havebeen given previously (Gilbert et al. (1996) J. Biol. Chem. 271,28271-28276).

[0224] Accurate mass determination of the enzymatic product of Cst-Ifrom Lac-FCHASE was consistent with the addition of sialic acid to theLac-FCHASE acceptor (FIG. 4). The product was identified as GM3-FCHASEsince the proton spectrum and ¹³C chemical shifts of the sugar moiety ofthe product (Table 6) were very similar to those for the GM3oligosaccharide or sialyllactose, (αNeu5Ac(2-3)βGal(I-4)pGlc; Sabesanand Paulson, supra.). The proton resonances of GM3-FCHASE were assignedfrom the COSY spectrum, the HSQC spectrum, and comparison of the protonand ¹³C chemical shifts with those ofαNeu5Ac(2-3)βGal(1-4)βGlcNAc-FCHASE (Gilbert et al., supra.). For thesetwo compounds, the proton and ¹³C chemical shifts for the Neu5Ac and Galresidues were within error bounds of each other (Id.). From a comparisonof the HSQC spectra of Lac-FCHASE and GM3-FCHASE, it is obvious that thelinkage site is at Gal C-3 due to the large downfield shift for Gal H-3and Gal C-3 upon sialylation typical for (2-3) sialyloligosaccarides(Sabesan and Paulson, supra.). Also, as seen before forαNeu5Ac(2-3)βGal(1-4)βGlcNAc-FCHASE (Gilbert et al., supra.), the NOEfrom H-3_(ax) of sialic acid to H-3 of Gal was observed typical of theαNeu5Ac(2-3)Gal linkage. TABLE 6 Comparison of the ¹³C chemical shiftsfor the FCHASE glycosides^(a) with those observed for lactose^(b)(Sabesan and Paulson, supra.), ganglioside oligosaccharides^(b) (Id.,Sabesan et al. (1984) Can. J. Chem. 62, 1034-1045) and (-8NeuAc2-)₃(Michon et al. (1987) Biochemistry 26, 8399-8405). The chemical shiftsat the glycosidation sites are underlined. Chemical Shift (ppm) ResidueC Lac- Lactose GM3- GM3OS GM2- GM2OS GM1a- GM1aOS GD3- 8NeuAc2 βGlc 1100.3  96.7 100.3  96.8 100.1  96.6 100.4  96.6 100.6  a 2 73.5 74.873.4 74.9 73.3 74.6 73.3 74.6 73.5 3 75.2 75.3 75.0 75.4 75.3 75.2 75.075.2 75.0 4 79.4 79.4 79.0 79.4 79.5 79.5 79.5 79.5 78.8 5 75.9 75.775.7 75.8 75.8 75.6 75.7 75.6 75.8 6 61.1 61.1 60.8 61.2 61.0 61.0 60.661.0 60.8 βGal(1-4) 1 104.1  103.8  103.6  103.7  103.6  103.5  103.6 103.5  103.6  b 2 72.0 71.9 70.3 70.4 71.0 70.9 70.9 70.9 70.3 3 73.573.5 76.4 76.6 75.3 75.6^(c) 75.1 75.2^(c) 76.3 4 69.7 69.5 68.4 68.578.3 78.0^(c) 78.1 78.0^(c) 68.5 5 76.4 76.3 76.0 76.2 75.0 74.9 74.975.0 76.1 6 62.1 62.0 62.1 62.0 62.2 61.4 62.0 61.5 62.0 αNeu5Ac 3 40.440.7 37.7 37.9 37.8 37.9 40.4 41.7 (2-3) 4 69.2 69.3 69.8 69.5 69.5 69.569.0  68.8^(d) c 5 52.6 52.7 52.7 52.5 52.6 52.5 53.0 53.2 6 73.7 73.974.0 73.9 73.8 73.9 74.9  74.5^(d) 7 69.0 69.2 69.0 68.8 69.0 68.9 70.370.0 8 72.6 72.8 73.3 73.1 73.1 73.1 79.1 79.1 9 63.4 63.7 63.9 63.763.7 63.7 62.5 62.1 NAc 22.9 23.1 23.2 22.9 23.3 22.9 23.2 23.2 βGalNAc1 103.8  103.6  103.4  103.4  (1-4) 2 53.2 53.2 52.0 52.0 d 3 72.3 72.281.4 81.2 4 68.8 68.7 68.9 68.8 5 75.6 75.2 75.1 75.2 6 61.8 62.0 61.562.0 NAc 23.2 23.5 23.4 23.5 βGal(1-3) 1 105.5  105.6  e 2 71.5 71.6 373.1 73.4 4 69.5 69.5 5 75.7 75.8 6 61.9 61.8 αNeu5Ac 3 41.2 41.2 (2-8)4 69.5 69.3 f 5 53.0 52.6 6 73.6 73.5 7 69.0 69.0 8 72.7 72.6 9 63.563.4 NAc 23.0 23.1

[0225] Accurate mass determination of the enzymatic product of Cst-IIfrom Lac-FCHASE indicated that two sialic acids had been added to theLac-FCHASE acceptor (FIG. 4). The proton resonances were assigned fromCOSY, 1D TOCSY and 1D NOESY and comparison of chemical shifts with knownstructures. The Glc H-1 to H-6 and Gal H-1 to H-4 resonances wereassigned from 1D TOCSY on the H-1 resonances. The Neu5Ac resonances wereassigned from COSY and confirmed by 1D NOESY. The 1D NOESY of the H-8,H-9-Neu5Ac resonances at 4.16 ppm was used to locate the H-9s and H-7resonances (Michon et al, supra.). The singlet appearance of the H-7resonance of Neu5Ac(2-3) arising from small vicinal coupling constantsis typical of the 2-8 linkage (Id.). The other resonances were assignedfrom the HSQC spectrum and ¹³C assignments for terminal sialic acid(Id.). The proton and ¹³C carbon chemical shifts of the Gal unit weresimilar to those in GM3-FCHASE, indicating the presence of theαNeu5Ac(2-3)Gal linkage. The J_(HH) values, proton and ¹³C chemicalshifts of the two sialic acids were similar to those ofαNeu5Ac(2-8)Neu5Ac in the α(2-8)-linked Neu5Ac trisaccharide (Sallowayet al. (1996) Infect. Immun. 64, 2945-2949) indicating the presence ofthat linkage. Hence, the product was identified as GD3-FCHASE.Sialylation at C-8 of Neu5Ac caused a downfield shift of −6.5 ppm in itsC-8 resonance from 72.6 ppm to 79.1 ppm.

[0226] The inter-residue NOEs for GD3-FCHASE were also typical of theαNeu5Ac(2-8)αNeu5Ac(2-3)βGal sequence. The largest inter-residue NOEsfrom the two H-3_(ax) resonances at 1.7-1.8 ppm of Neu5Ac(2-3) andNeu5Ac(2-8) are to the Gal H-3 and -8)Neu5Ac H-8 resonances. Smallerinter-residue NOEs to Gal H-4 and -8)Neu5Ac H-7 are also observed. NOEson FCHASE resonances are also observed due the overlap of an FCHASEresonance with the H-3_(ax) resonances (Gilbert et al., supra.). Theinter-residue NOE from H-³ _(eq) of Neu5Ac(2-3) to Gal H-3 is alsoobserved. Also, the intra-residues confirmed the proton assignments. TheNOEs for the 2-8 linkage are the same as those observed for the-8Neu5Acα2-polysaccharide (Michon et al., supra.).

[0227] The sialic acid glycosidic linkages could also be confirmed bythe use of the HMBC experiment which detects ³J(C, H) correlationsacross the glycosidic bond. The results for both α-2,3 and α-2,8linkages indicate the ³J(C, H) correlations between the two Neu5Acanomeric C-2 resonances and Gal H-3 and -8)Neu5Ac H-8 resonances. Theintra-residue correlations to the H-3_(ax) and H-³ _(eq) resonances ofthe two Neu5Ac residues were also observed. The Glc (C-1, H-2)correlation is also observed since there was partial overlap of thecrosspeaks at 101 ppm with the crosspeaks at 100.6 ppm in the HMBCspectrum.

[0228] Accurate mass determination of the enzymatic product of CgtA fromGM3-FCHASE indicated that a N-acetylated hexose unit had been added tothe GM3-FCHASE acceptor (FIG. 4). The product was identified asGM2-FCHASE since the glycoside proton and ¹³C chemical shifts weresimilar to those for GM2 oligosaccharide (GM2OS) (Sabesan et al. (1984)Can. J. Chem. 62, 1034-1045). From the HSQC spectrum for GM2-FCHASE andthe integration of its proton spectrum, there are now two resonances at4.17 ppm and 4.18 ppm along with a new anomeric “d1” and two NAc groupsat 2.04 ppm. From TOCSY and NOESY experiments, the resonance at 4.18 ppmwas unambiguously assigned to Gal H-3 because of the strong NOE betweenH-1 and H-3. For βgalactopyranose, strong intra-residue NOEs between H-1and H-3 and H-1 and H-5 are observed due to the axial position of theprotons and their short interproton distances (Pavliak et al. (1993) J.Biol. Chem. 268, 14146-14152; Brisson et al. (1997) Biochemistry 36,3278-3292; Sabesan et al. (1984) Can. J. Chem. 62, 1034-1045). From theTOCSY spectrum and comparison of the H1 chemical shifts of GM2-FCHASEand GM2OS (Sabesan et al., supra.) the resonance at 4.17 ppm is assignedas Gal H-4. Similarly, from TOCSY and NOESY spectra, the H-1 to H-5 ofGalNAc and Glc, and H-3 to H-6 of Neu5Ac were assigned. Due to broadlines, the multiplet pattern of the resonances could not be observed.The other resonances were assigned from comparison with the HSQCspectrum of the precursor and ¹³C assignments for GM2OS (Sabesan et al.,supra.). By comparing the HSQC spectra for GM3- and GM2-FCHASEglycosides, a −9.9 ppm downfield shift between the precursor and theproduct occurred on the Gal C-4 resonance. Along with intra-residue NOEsto H-3 and H-5 of GalNAc, the inter-residue NOE from GalNAc H-1 to GalH-4 at 4.17 ppm was also observed confirming the βGalNAc(1-4)Galsequence. The observed NOEs were those expected from the conformationalproperties of the GM2 ganglioside (Sabesan et al., supra.).

[0229] Accurate mass determination of the enzymatic product of CgtB fromGM2-FCHASE indicated that a hexose unit had been added to the GM2-FCHASEacceptor (FIG. 4). The product was identified as GM1a-FCHASE since theglycoside ¹³C chemical shifts were similar to those for the GM1aoligosaccharide (Id.). The proton resonances were assigned from COSY, 1DTOCSY and 1D NOESY. From a 1D TOCSY on the additional “e1” resonance ofthe product, four resonances with a mutltiplet pattern typical ofβ-galactopyranose were observed. From a 1D TOCSY and 1D NOESY on the H-1resonances of βGalNAc, the H-1 to H-5 resonances were assigned. TheβGalNAc H-1 to H-4 multiplet pattern was typical of theβ-galactopyranosyl configuration, confirming the identity of this sugarfor GM2-FCHASE. It was clear that upon glylcosidation, the majorperturbations occurred for the βGalNAc resonances, and there was −9.1ppm downfield shift between the acceptor and the product on the GalNAcC-3 resonance. Also, along with intra-residue NOEs to H-3, H-5 of Gal,an inter-residue NOE from Gal H-1 to GalNAc H-3 and a smaller one toGalNAc H-4 were observed, confirming the βGal(1-3)GalNAc sequence. Theobserved NOEs were those expected from the conformational properties ofthe GM1a ganglioside (Sabesan et al., supra.).

[0230] There was some discrepancy with the assignment of the C-3 and C-4βGal(1-4) resonances in GM2OS and GM1OS which are reversed from thepublished data (Sabesan et al., supra.). Previously, the assignmentswere based on comparison of ¹³C chemical shifts with known compounds.For GM1a-FCHASE, the assignment for H-3 of Gal(1-4) was confirmed byobserving its large vicinal coupling, J_(2,3)=10 Hz, directly in theHSQC spectrum processed with 2 Hz/point in the proton dimension. The H-4multiplet is much narrower (<5 Hz) due to the equatorial position of H-4in galactose (Sabesan et al., supra.). In Table 6, the C-4 and C-6assignments of one of the sialic acids in (-8Neu5Ac2-)₃ also had to bereversed (Michon et al., supra.) as confirmed from the assignments ofH-4 and H-6.

[0231] The ¹³C chemical shifts of the FCHASE glycosides obtained fromHSQC spectra were in excellent agreement with those of the referenceoligosaccharides shown in Table 6. Differences of over 1 ppm wereobserved for some resonances and these are due to different aglycons atthe reducing end. Excluding these resonances, the averages of thedifferences in chemical shifts between the FCHASE glycosides and theirreference compound were less than ±0.2 ppm. Hence, comparison of protonchemical shifts, J_(HH) values and ¹³C chemical shifts with knownstructures, and use of NOEs or HMBC were all used to determine thelinkage specificity for various glycosyltransferases. The advantage ofusing HSQC spectra is that the proton assignment can be verifiedindependently to confirm the assignment of the ¹³C resonances of theatoms at the linkage site. In terms of sensitivity, the proton NOEs arethe most sensitive, followed by HSQC then HMBC. Using a nano-NMR probeinstead of a 5 mm NMR probe on the same amount of material reducedconsiderably the total acquisition time, making possible the acquisitionof an HMBC experiment overnight.

[0232] Discussion

[0233] In order to clone the LOS glycosyltransferases from C. jejuni, weemployed an activity screening strategy similar to that which wepreviously used to clone the α-2,3-sialyltransferase from Neisseriameningitidis (Gilbert et al., supra.). The activity screening strategyyielded two clones which encoded two versions of the sameα-2,3-sialyltransferase gene (cst-I). ORF analysis suggested that a 430residue polypeptide is responsible for the α-2,3-sialyltransferaseactivity. To identify other genes involved in LOS biosynthesis, wecompared a LOS biosynthesis locus in the complete genome sequence of C.jejuni NCTC 11168 to the corresponding locus from C. jejuni OH4384.Complete open reading frames were identified and analyzed. Several ofthe open reading frames were expressed individually in E. coli,including a β-1,4-N-acetylgalactosaminyl-transferase (cgtA), aβ-1,3-galactosyltransferase (cgtB) and a bifunctional sialyltransferase(cst-II).

[0234] The in vitro synthesis of fluorescent derivatives of nanomoleamounts of ganglioside mimics and their NMR analysis confirmunequivocally the linkage specificity of the four clonedglycosyltransferases. Based on these data, we suggest that the pathwaydescribed in FIG. 4 is used by C. jejuni OH4384 to synthesize a GT1amimic. This role for cgtA is further supported by the fact that C.jejuni OH4342, which carries an inactive version of this gene, does nothave β-1,4-GalNAc in its LOS outer core (FIG. 1). The cst-II gene fromC. jejuni OH4384 exhibited both α-2,3- and α-2,8-sialyltransferase in anin vitro assay while cst-II from C. jejuni O:19 (serostrain) showed onlyα-2,3-sialyltransferase activity (Table 5). This is consistent with arole for cst-II in the addition of a terminal α-2,8-linked sialic acidin C. jejuni OH4382 and OH4384, both of which have identical cst-IIgenes, but not in C. jejuni O:19 (serostrain, see FIG. 1). There are 8amino acid differences between the Cst-II homologues from C. jejuni O:19(serostrain) and OH4382/84.

[0235] The bifunctionality of cst-II might have an impact on the outcomeof the C. jejuni infection since it has been suggested that theexpression of the terminal di-sialylated epitope might be involved inthe development of neuropathic complications such as the Guillain-Barrésyndrome (Salloway et al. (1996) Infect. Immun. 64, 2945-2949). It isalso worth noting that its bifunctional activity is novel among thesialyltransferases described so far. However, a bifunctionalglycosyltransferase activity has been described for the3-deoxy-D-manno-octulosonic acid transferase from E. coli (Belunis, C.J., and Raetz, C. R. (1992) J. Biol. Chem. 267, 9988-9997).

[0236] The mono/bi-functional activity of cst-II and theactivation/inactivation of cgtA seem to be two forms of phase variationmechanisms that allow C. jejuni to make different surface carbohydratesthat are presented to the host. In addition to those small genealterations that are found among the three O:19 strains (serostrain,OH4382 and OH4384), there are major genetic rearrangements when the lociare compared between C. jejuni OH4384 and NCTC 11168 (an O:2 strain).Except for the prB gene, the cst-I locus (including cysN and cysD) isfound only in C. jejuni OH4384. There are significant differences in theorganization of the LOS biosynthesis locus between strains OH4384 andNCTC 11168. Some of the genes are well conserved, some of them arepoorly conserved while others are unique to one or the other strain. Twogenes that are present as separate ORFs (#5a: cgtA and #10a: NeuA) inOH4384 are found as an in-frame fusion ORF in NCTC 11168 (ORF #5b/#10b).β-N-acetylgalactosaminyltransferase activity was detected in thisstrain, which suggests that at least the cgtA part of the fusion may beactive.

[0237] In summary, this Example describes the identification of severalopen reading frames that encode enzymes involved in the synthesis oflipooligosaccharides in Campylobacter.

[0238] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference for all purposes.

1 49 1 11474 DNA Campylobacter jejuni 11.5 kb PCR product from C. jejuniOH4384 including LOS biosynthesis locus 1 aaagaatacg aatttgctaaagaggtttta aatcttagtg gtattgatga aacacatata 60 gaattagcgc caaaatttaatcttgaagag ctaatggctt ttacaaaaat gatggatctt 120 atcataggaa atgatagcggtccaacacat ttagcttttg ctttaaataa agcatctatt 180 acgatttttg gtgcaacaccaagctaccgc aatgcttttc aaactcatat caataaaatc 240 attgatacag gtaaaaaaatccaaaatgcc aagcatatcg ataaaagtga tttttgtatc 300 acgcgtatag aagaagaagatatcttcaaa cttgccaaag gcttacttaa tgaaaaatag 360 tgatagaata tatcttagtctttattatat tttgaaattt tttgttactt ttatgcctga 420 ttgtatcttg cattttttagctttgattgt agcaagaatc gcttttcatc ttaacaaaaa 480 acaccgcaaa atcatcaatacaaatttgca aatctgtttt cctcaataca ctcaaaaaga 540 acgcgataaa ttgtctttaaaaatttatga aaattttgct caatttggga ttgattgttt 600 gcaaaatcaa aacaccaccaaagaaaaaat tctcaataaa gtaaatttca tcaatgaaaa 660 ttttcttata gatgccctggctttaaagcg tcctattatc ttcacaactg cacactatgg 720 aaactgggaa attttaagccttgcttatgc ggctaaatat ggtgcgattt ccatagtggg 780 aaaaaagtta aaaagtgaagttatgtatga aattttaagc caaagtcgca cccaatttga 840 catagaactt attgacaaaaaaggcggtat aagacaaatg ctaagtgctc taaaaaagga 900 gagagctttg ggaattttaactgatcaaga ctgcgtagaa aacgaaagcg taagattaaa 960 attttttaac aaagaagtgaattatcaaat gggagcaagc cttatcgcac aaagaagcaa 1020 tgctttgatc atccctgtttatgcctataa agaaggtggt aaattttgca tagagttttt 1080 taaagcaaaa gattctcaaaatgcaagttt agaagaactg acactttatc aagcacaaag 1140 ttgcgaagaa atgattaaaaaaagaccttg ggaatacttt ttttttcata gacgctttgc 1200 tagttataat gaggaaatttacaagggtgc aaaatgaatc taaaacaaat aagcgttatt 1260 atcatcgtaa aaaatgctgagcaaactttg cttgagtgtt taaattcttt aaaagatttt 1320 gatgaaatta ttttacttaacaatgaaagt agcgataata ccctaaaaat agctaatgaa 1380 tttaaaaaag attttgctaatttatatatt tatcacaatg cttttatagg ttttggagct 1440 ttaaaaaatc ttgctttaagttatgcaaaa aatgattgga ttttaagcat tgatgctgat 1500 gaagtgcttg aaaatgagtgtattaaagag cttaaaaatt taaaacttca agaagataat 1560 atcatcgcac ttagccgtaaaaatctctat aaaggcgaat ggataaaggc atgtggttgg 1620 tggcctgatt atgttttgagaatttttaat aaaaatttca ctcgttttaa tgataattta 1680 gtacatgaaa gccttgttttgccaagtaat gctaaaaaaa tttatcttaa aaatggattg 1740 aagcattatt cttataaggatatctctcac ttaattgaca aaatgcagta ctactcaagt 1800 ctttgggcaa aacaaaatatacacaaaaaa agtggtgttt taaaagcaaa tttaagagct 1860 ttttggactt tttttagaaattatttttta aaaaatggct ttttatatgg ttataagggt 1920 tttataatta gcgtttgttctgcattggga acatttttta aatatatgaa attatatgaa 1980 cttcaaagac aaaaaccaaaaacttgcgct ttaataataa taacttataa tcaaaaagaa 2040 cgccttaaac tagtgcttgatagtgttaaa aatctagcct ttttacccaa tgaagtttta 2100 atcgcagatg atggtagcaaagaagataca gcaaggctta ttgaagaata tcaaaaagat 2160 tttccttgtc ctttaaaacacatttggcaa gaagatgaag ggtttaaact tagtaaaagt 2220 cgcaacaaaa ctataaaaaacgctgatagt gaatatataa tagttattga tggtgatatg 2280 attttggaaa aagatttcataaaagaacat ttagaatttg cacaaagaaa gcttttttta 2340 caaggttcaa gagtaattttaaataaaaaa gaaagcgaag aaattttaaa caaagatgat 2400 tatcgcataa tttttaataaaaaagatttt aaaagttcta aaaattcttt tttagctaaa 2460 atattttaca gtctttcaaaaaaaagatga aaaaatcttt taaaaaacca ctcttattaa 2520 aggtattagg ggttgcaatatgagtttttt taaaactgat tttgatgaac ttgatggttt 2580 taatgaaaat tttattggttggggtagaga agatagtgaa tttgttgcta gatttttatt 2640 taataaaggc atttttagacgattaaaatt taaagctatt gcttatcata tttatcacaa 2700 agaaaatagc aaaaaaatgcttgaaagcaa tcatcaaatt tatttagata ccatcaaaaa 2760 taaaaagatt tcttggagataaaacatgaa gaaaataggt gtagttatac caatctataa 2820 tgtagaaaaa tatttaagagaatgtttaga tagcgttatc aatcaaactt atactaactt 2880 agaaatcata cttgtcaatgatggtagcac agatgaacac tcactcaata ttgcaaaaga 2940 atatacctta aaagataaaagaataactct ttttgataag aaaaatgggg gtttaagttc 3000 agctagaaat ataggtatagaatactttag cggggaatat aaattaaaaa acaaaactca 3060 acatataaaa gaaaattctttaatagaatt tcaattggat ggtaataatc cttataatat 3120 atataaagca tataaaagctctcaagcttt taataatgaa aaagatttaa ccaattttac 3180 ttaccctagt atagattatattatattctt agatagtgat aattattgga aactaaactg 3240 catagaagaa tgcgttataagaatgaaaaa tgtggatgta ttgtggtttg accatgattg 3300 cacctatgaa gacaatataaaaaataagca caaaaaaaca aggatggaaa tttttgattt 3360 taaaaaagaa tgtataatcactccaaaaga atatgcaaat cgagcattaa gtgtaggatc 3420 tagagatatt tcttttggatggaatggaat gattgatttt aattttttaa agcaaattaa 3480 acttaaattt ataaattttattatcaatga agatatacac tttgggataa ttttgtttgc 3540 tagtgctaat aaaatttatgttttatcaca aaagttgtat ttgtgtcgtt taagagcaaa 3600 cagtatatca aatcatgataagaagattac aaaagcaaat gtgtcagagt attttaaaga 3660 tatatatgaa actttcggggaaaacgctaa ggaagcaaaa aattatttaa aagcagcaag 3720 cagggttata actgctttaaaattgataga attttttaaa gatcaaaaaa acgaaaatgc 3780 acttgctata aaagaaacatttttaccttg ctatgccaaa aaagctttaa tgattaaaaa 3840 atttaaaaaa gatcctttaaatttaaagga acaattagtt ttaattaaac cttttattca 3900 aacaaaactt ccttatgatatttggaaatt ttggcaaaaa ataaaaaata tttaataata 3960 aaaatataaa aaattaattaatttttaggt ataatcacta taattatagg agaaaatatt 4020 ttatatgcta tttcaatcatactttgtgaa aataatttgc ttattcatcc cttttagaaa 4080 aattagacat aaaataaaaaaaacattttt actaaaaaac atacaacgag ataaaatcga 4140 ttcttattta ccaaaaaaaactcttgtgca aattaataaa tacaacaatg aagatttaat 4200 taaacttaat aaagctattataggggaggg gcataaagga tattttaatt atgatgaaaa 4260 atctaaagat ccaaaatctcctttgaatcc ttgggctttt atacgagtaa aaaatgaagc 4320 tattacctta aaagcttctcttgaaagcat attgcctgct atccaaagag gtgttatagg 4380 atataatgat tgtaccgatggaagtgaaga aataattcta gaattttgca aacaatatcc 4440 ttcatttata ccaataaaatatccttatga aattcaaatt caaaacccaa aatcagaaga 4500 aaataaactc tatagctattataattatgt tgcaagtttt ataccaaaag atgagtggct 4560 tataaaaata gatgtggatcatatctatga tgctaaaaaa ctttataaaa gcttctatat 4620 accaaaaaac aaatatgatgtagttagtta ttcaagggtt gatattcact attttaatga 4680 taattttttt ctttgtaaagataataatgg caatatattg aaagaaccag gagattgctt 4740 gcttatcaat aattataacttaaaatggaa agaagtatta attgacagaa tcaataacaa 4800 ttggaaaaaa gcaacaaaacaaagtttttc ttcaaatata cactctttag agcaattaaa 4860 gtataaacac aggatattatttcacactga attaaataat tatcattttc cttttttaaa 4920 aaaacataga gctcaagatatttataaata taattggata agtattgaag aatttaaaaa 4980 attctattta caaaatattaatcataaaat agaaccttct atgatttcaa aagaaactct 5040 aaaaaaaata ttcttaacattgttttaaaa attttttata tttaaataaa atttttaaag 5100 ttaaaatatt tattttagctaataatgtaa ccattaattt tgttcttttt attttatata 5160 tttgaatata tagcaaatatttaattagca catagagaac gctacaatac ttgtttaaaa 5220 tataattttg ccttaaatagtttaaaacca actgcaactc ttgaatatta tttttaacaa 5280 gcacttcatt cttagtattacaaattgaat tattattagg cacgtaatga tataaattac 5340 agttcatata tgctattttttgagcttgac ttaacattgg ataatataac aatacatctt 5400 cagccatatt gattttaacatctttctcga gtcttaaact cgcaaaagct tctaaataca 5460 atttctttct tataagtttcccccacatag tccaatataa atttttcttt gcaataattt 5520 tttttacaaa ctcttttttgctataaaaac cagaattaaa gtcaaacttt ttatatgaaa 5580 taacattact ttcaacaatagcattgaaaa acactaaatc aacttcatcc tgttcatcta 5640 aaatttttat acactcttcacaagcattta gttccaaata atcatcagga tctaaaaaca 5700 ttatataagg agagtttgctactttcacac cttcatatct tgctcttaaa agacctaagt 5760 ttttttcatt gtggattatttttattcttt tgtctttttt agagtattct ttggctatat 5820 ttatactatt atcatttccacaatcatcaa ctacaattat ttctatatct ttaaaagtct 5880 gattgataca gctttctattgcccttgcta tatattgttc cacattataa gttggtaaga 5940 tgattgaaat tttaaacatatttattcctt attttattat aatttaatta taacataaaa 6000 tctattttga taaaatcgttaaaaataaat cttgatggaa aataatcatg aaaaaagtta 6060 ttattgctgg aaatggaccaagtttaaaag aaattgatta ttcaagacta ccaaatgatt 6120 ttgatgtatt tagatgtaatcaattttatt ttgaagataa atactatctt ggtaaaaaat 6180 gcaaggcagt attttacaatcctattcttt tttttgaaca atactacact ttaaaacatt 6240 taatccaaaa tcaagaatatgagaccgaac taattatgtg ttctaattac aaccaagctc 6300 atctagaaaa tgaaaattttgtaaaaactt tttacgatta ttttcctgat gctcatttgg 6360 gatatgattt tttcaaacaacttaaagatt ttaatgctta ttttaaattt cacgaaattt 6420 atttcaatca aagaattacctcaggggtct atatgtgtgc agtagccata gccctaggat 6480 acaaagaaat ttatctttcgggaattgatt tttatcaaaa tgggtcatct tatgcttttg 6540 atactaaaca aaaaaatcttttaaaattgg ctcctaattt taaaaatgat aattcacact 6600 atatcggaca tagtaaaaatacagatataa aagctttaga atttctagaa aaaacttaca 6660 aaataaaact atattgcttatgtcctaaca gtcttttagc aaattttata gaactagcgc 6720 caaatttaaa ttcaaattttatcatacaag aaaaaaataa ctacactaaa gatatactca 6780 taccttctag tgaggcttatggaaaatttt caaaaaatat taattttaaa aaaataaaaa 6840 ttaaagaaaa tatttattacaagttgataa aagatctatt aagattacct agtgatataa 6900 agcattattt caaaggaaaataaatgaaag aaataaaaat acaaaatata atcataagtg 6960 aagaaaaagc acccttagtcgtgcctgaaa taggcattaa tcataatggc agtttagaac 7020 tagctaaaat tatggtagatgcagccttta gcacaggtgc taagattata aagcatcaaa 7080 cccacatcgt tgaagatgagatgagtaagg ccgctaaaaa agtaattcct ggtaatgcaa 7140 aaataagcat ttatgagattatgcaaaaat gtgctttaga ttataaagat gagctagcac 7200 ttaaagaata cacagaaaaattaggtcttg tttatcttag cacacctttt tctcgtgcag 7260 gtgcaaaccg cttagaagatatgggagtta gtgcttttaa gattggttca ggtgagtgta 7320 ataattatcc gcttattaaacacatagcag cctttaaaaa gcctatgata gttagcacag 7380 ggatgaatag tattgaaagtataaaaccaa ctgtaaaaat cttattagac aatgaaattc 7440 cctttgtttt aatgcacacaaccaatcttt acccaacccc gcataatctt gtaagattaa 7500 acgctatgct tgaattaaaaaaagaatttt cttgtatggt aggcttaagc gaccacacaa 7560 cagataatct tgcgtgtttaggtgcggttg cacttggtgc ttgtgtgctt gaaagacatt 7620 ttactgatag tatgcatagaagtggccctg atatagtttg ttctatggat acacaggctt 7680 taaaagagct tattatacaaagtgagcaaa tggctataat gagaggaaat aatgaaagta 7740 aaaaagcagc taagcaagagcaagtcacaa ttgattttgc ctttgcaagc gtagtcagca 7800 ttaaagatat taaaaaaggcgaagttttat ctatggataa tatttgggtt aaaagacctg 7860 gacttggtgg aattagtgcagctgaatttg aaaatatttt aggcaaaaaa gcattaagag 7920 atatagaaaa tgatactcagttaagctatg aggattttgc gtgaaaaaaa tcctttttat 7980 aacaggcact agggctgattattctaagat taaatcttta atgtacaggg tgcaaaactc 8040 aagcgaattt gaactttacatctttgcaac aggaatgcac ttaagcaaaa attttggcta 8100 tacagttaaa gaactttataaaaatggctt taaaaatatt tatgaattta taaattacga 8160 taaatatttt tcaaccgataaggctttagc cactacaatt gatggatttt caagatatgt 8220 aaatgagcta aaacctgatttaatcgtagt acatggagat agaatcgagc ctttagcagc 8280 agctattgtt ggagcattaaacaatatctt agtagcacat attgaaggtg gagagatttc 8340 aggaactatt gatgatagcttacgccacgc tatatcaaaa ctagcacata ttcatttagt 8400 aaatgatgag tttgcaaaaaggcgtttaat gcagcttgga gaagatgaaa aatctatttt 8460 tatcataggt tcgcctgatttagaactttt aaacgataat aaaatttcac ttaatgaagc 8520 aaaaaaatat tatgatataaattatgaaaa ctacgctttg cttatgtttc atcctgttac 8580 aactgaaatt acaagcattaaaaatcaagc agataattta gtaaaagcac tgatacaaag 8640 taacaaaaat tatattgttatttatccaaa taatgattta ggttttgaat taatcttgca 8700 aagctatgaa gaacttaaaaataaccctag atttaagctt tttccatcgc ttagatttga 8760 gtattttata actttgttaaaaaatgctga ttttataata ggtaattcaa gttgtatttt 8820 aaaagaggcc ttatacttaaaaacagcagg aattttagtt ggctcaaggc aaaatggaag 8880 acttggcaat gaaaatacactaaaagttaa tgcaaatagt gatgaaatac taaaagctat 8940 taataccatt cataaaaaacaagatttatt tagcgccaag ttagagattt tagatagctc 9000 aaaattattt tttgaatatttacaaagcgg agaatttttt aaacttaaca cacaaaaagt 9060 ttttaaggat ataaaatgagcttagcaata atccctgctc gtggtggctc aaagggtatt 9120 aaaaataaaa atttggttttattaaacaat aaacctttaa tttattacac cattaaagct 9180 gcactaaata ctaaaagcattagtaaagtt gttgtaagca gtgatagtga tgaaatttta 9240 aattatgcaa aaagtcaaaatgttgatatt ttaaaacgcc caattagcct tgcacaagat 9300 aatactacaa gcgataaagtgcttttacat gctctaaaat tttacaaaga ttatgaagat 9360 gtagtttttt tacaacccacttcgccgcta agaacaaata ttcatattga tgaggctttt 9420 aatctttata aaaatagcaatgcaaatgcc ctaattagcg taagcgaatg tgataataaa 9480 attctaaaag cctttgtttgtaatgaatat ggcgatttag cagggatttg taatgatgaa 9540 tatcctttta tgccaaggcaaaaattgcct aaaacatata tgagcaatgg tgcaatttat 9600 attttaaaga taaaagaatttttaaacaat cctagctttt tacaaagcaa aaccaagcat 9660 tttttaatgg atgaaagctcaagtttagat attgactgtt tggaggattt aaaaaaggct 9720 gaacagatat ggaaaaaataaccttaaaat gcaataaaaa tatattaaat ttattaaagc 9780 aatataatat ttatacaaaaacttatatag aaaatcctag aagattttca agactaaaaa 9840 ccaaagattt tataacctttccattggaaa acaatcaact agagagtgta gcggggctgg 9900 ggatagaaga atattgtgcttttaaattta gcaatatctt acatgaaatg gattcatttt 9960 cttttagcgg atcttttctacctcattata caaaagttgg aaggtattgt tcaatttctg 10020 atggggtttc tatgtttaactttcaacatc ctatggatag aatcagcact gcaagtttta 10080 cctatgaaac aaatcatagttttattaacg atgcttgcca aaatcacatc aacaaaacat 10140 ttcctatagt taaccataatccaagctcat caataacgca tttaattata caagatgatg 10200 tttggatagg aaaagatgttttgcttaaac agggtatcac acttgggact ggatgtgtca 10260 taggacaaag agctgtagttactaaagatg taccacctta tgctatagtt gcaggaattc 10320 cagccaaaat tatcaaatatagatttgatg aaaaaacaat agaaagatta ttaaaaattc 10380 aatggtggaa atatcattttgctgattttt atgatattga tcttaattta aaaataaacc 10440 aatatcttga cctactagaagaaaaaatca taaaaaaatc aatttcctac tataatccaa 10500 ataaacttta ttttagagatattttagaac taaaatcaaa aaaaattttt aatctatttt 10560 aatctatttt tcacccctgcttcctctctc tttaaaactt caaataattt ctgatgaaat 10620 tcatcatgtg caaactctttggatagtttt tttatgattt cattactttt ctttttatca 10680 tgataatttt gatttaaaatttctttattt ttattctcat atcttccatt tggattaaat 10740 tcataatgat aaatgcaagttttaaaaaca gctattttct cacaaaacat aaaataaata 10800 taacaaaaaa gcacatcttcgccataattc aaacgctcat ctattttaat tttttcaaaa 10860 ctttttaaga tgatatcttttttaaagcac ttcgcccaaa ccgaccagca aaaatgcctt 10920 tgtttgctta aaaattctaaaaattccttt tgattaaaaa cttcatcttg tttaaaacga 10980 taaaattgtt tggtttttaccctatgcaca aaggcatcaa aacaaagcaa atcaaaacct 11040 tttttcatct ctttaaacgctatttcacaa gcatcaggtg ttaaaaaatc atcactatct 11100 aaaaacatta taaaatcagaactagaatgc aaaaccccca aatttctact tgcaaaagtg 11160 cctaaatttt cttcattttgaaagattttt attcttggat ctttttttgc aaattctaaa 11220 accatattta aactattatctttactttta tcatcgataa tcaaaatttc aatatctttt 11280 aaagtctgat ttatacaactttgcaaagct cttgagataa aatcgcaaga attaaaaagc 11340 gggattatga tagaaagttgtggcatattt ttcctaaatt ttgttaaaat aataaaaaca 11400 attctatcaa agtttaggaaatttatgaaa atttttatac accttccaac ctggttaggc 11460 gatacggtaa tggc 114742 876 DNA Campylobacter jejuni CDS (1)..(876) bifunctionalalpha-2,3/alpha 2,8-sialyltransferase Campylobacter sialyltransferase II(cstII) from C. jejuni strain OH4384 (ORF 7a of lipooligosaccharide(LOS) biosynthesis locus) 2 atg aaa aaa gtt att att gct gga aat gga ccaagt tta aaa gaa att 48 Met Lys Lys Val Ile Ile Ala Gly Asn Gly Pro SerLeu Lys Glu Ile 1 5 10 15 gat tat tca aga cta cca aat gat ttt gat gtattt aga tgt aat caa 96 Asp Tyr Ser Arg Leu Pro Asn Asp Phe Asp Val PheArg Cys Asn Gln 20 25 30 ttt tat ttt gaa gat aaa tac tat ctt ggt aaa aaatgc aag gca gta 144 Phe Tyr Phe Glu Asp Lys Tyr Tyr Leu Gly Lys Lys CysLys Ala Val 35 40 45 ttt tac aat cct att ctt ttt ttt gaa caa tac tac acttta aaa cat 192 Phe Tyr Asn Pro Ile Leu Phe Phe Glu Gln Tyr Tyr Thr LeuLys His 50 55 60 tta atc caa aat caa gaa tat gag acc gaa cta att atg tgttct aat 240 Leu Ile Gln Asn Gln Glu Tyr Glu Thr Glu Leu Ile Met Cys SerAsn 65 70 75 80 tac aac caa gct cat cta gaa aat gaa aat ttt gta aaa actttt tac 288 Tyr Asn Gln Ala His Leu Glu Asn Glu Asn Phe Val Lys Thr PheTyr 85 90 95 gat tat ttt cct gat gct cat ttg gga tat gat ttt ttc aaa caactt 336 Asp Tyr Phe Pro Asp Ala His Leu Gly Tyr Asp Phe Phe Lys Gln Leu100 105 110 aaa gat ttt aat gct tat ttt aaa ttt cac gaa att tat ttc aatcaa 384 Lys Asp Phe Asn Ala Tyr Phe Lys Phe His Glu Ile Tyr Phe Asn Gln115 120 125 aga att acc tca ggg gtt tat atg tgt gca gta gcc ata gcc ctagga 432 Arg Ile Thr Ser Gly Val Tyr Met Cys Ala Val Ala Ile Ala Leu Gly130 135 140 tac aaa gaa att tat ctt tcg gga att gat ttt tat caa aat gggtca 480 Tyr Lys Glu Ile Tyr Leu Ser Gly Ile Asp Phe Tyr Gln Asn Gly Ser145 150 155 160 tct tat gct ttt gat act aaa caa aaa aat ctt tta aaa ttggct cct 528 Ser Tyr Ala Phe Asp Thr Lys Gln Lys Asn Leu Leu Lys Leu AlaPro 165 170 175 aat ttt aaa aat gat aat tca cac tat atc gga cat agt aaaaat aca 576 Asn Phe Lys Asn Asp Asn Ser His Tyr Ile Gly His Ser Lys AsnThr 180 185 190 gat ata aaa gct tta gaa ttt cta gaa aaa act tac aaa ataaaa cta 624 Asp Ile Lys Ala Leu Glu Phe Leu Glu Lys Thr Tyr Lys Ile LysLeu 195 200 205 tat tgc tta tgt cct aac agt ctt tta gca aat ttt ata gaacta gcg 672 Tyr Cys Leu Cys Pro Asn Ser Leu Leu Ala Asn Phe Ile Glu LeuAla 210 215 220 cca aat tta aat tca aat ttt atc ata caa gaa aaa aat aactac act 720 Pro Asn Leu Asn Ser Asn Phe Ile Ile Gln Glu Lys Asn Asn TyrThr 225 230 235 240 aaa gat ata ctc ata cct tct agt gag gct tat gga aaattt tca aaa 768 Lys Asp Ile Leu Ile Pro Ser Ser Glu Ala Tyr Gly Lys PheSer Lys 245 250 255 aat att aat ttt aaa aaa ata aaa att aaa gaa aat atttat tac aag 816 Asn Ile Asn Phe Lys Lys Ile Lys Ile Lys Glu Asn Ile TyrTyr Lys 260 265 270 ttg ata aaa gat cta tta aga tta cct agt gat ata aagcat tat ttc 864 Leu Ile Lys Asp Leu Leu Arg Leu Pro Ser Asp Ile Lys HisTyr Phe 275 280 285 aaa gga aaa taa 876 Lys Gly Lys 290 3 291 PRTCampylobacter jejuni bifunctional alpha-2,3/alpha 2,8-sialyltransferaseCampylobacter sialyltransferase II (cstII) from C. jejuni strain OH4384(ORF 7a of lipooligosaccharide (LOS) biosynthesis locus) 3 Met Lys LysVal Ile Ile Ala Gly Asn Gly Pro Ser Leu Lys Glu Ile 1 5 10 15 Asp TyrSer Arg Leu Pro Asn Asp Phe Asp Val Phe Arg Cys Asn Gln 20 25 30 Phe TyrPhe Glu Asp Lys Tyr Tyr Leu Gly Lys Lys Cys Lys Ala Val 35 40 45 Phe TyrAsn Pro Ile Leu Phe Phe Glu Gln Tyr Tyr Thr Leu Lys His 50 55 60 Leu IleGln Asn Gln Glu Tyr Glu Thr Glu Leu Ile Met Cys Ser Asn 65 70 75 80 TyrAsn Gln Ala His Leu Glu Asn Glu Asn Phe Val Lys Thr Phe Tyr 85 90 95 AspTyr Phe Pro Asp Ala His Leu Gly Tyr Asp Phe Phe Lys Gln Leu 100 105 110Lys Asp Phe Asn Ala Tyr Phe Lys Phe His Glu Ile Tyr Phe Asn Gln 115 120125 Arg Ile Thr Ser Gly Val Tyr Met Cys Ala Val Ala Ile Ala Leu Gly 130135 140 Tyr Lys Glu Ile Tyr Leu Ser Gly Ile Asp Phe Tyr Gln Asn Gly Ser145 150 155 160 Ser Tyr Ala Phe Asp Thr Lys Gln Lys Asn Leu Leu Lys LeuAla Pro 165 170 175 Asn Phe Lys Asn Asp Asn Ser His Tyr Ile Gly His SerLys Asn Thr 180 185 190 Asp Ile Lys Ala Leu Glu Phe Leu Glu Lys Thr TyrLys Ile Lys Leu 195 200 205 Tyr Cys Leu Cys Pro Asn Ser Leu Leu Ala AsnPhe Ile Glu Leu Ala 210 215 220 Pro Asn Leu Asn Ser Asn Phe Ile Ile GlnGlu Lys Asn Asn Tyr Thr 225 230 235 240 Lys Asp Ile Leu Ile Pro Ser SerGlu Ala Tyr Gly Lys Phe Ser Lys 245 250 255 Asn Ile Asn Phe Lys Lys IleLys Ile Lys Glu Asn Ile Tyr Tyr Lys 260 265 270 Leu Ile Lys Asp Leu LeuArg Leu Pro Ser Asp Ile Lys His Tyr Phe 275 280 285 Lys Gly Lys 290 4876 DNA Campylobacter jejuni CDS (1)..(876) bifunctional alpha-2,3/alpha2,8-sialyltransferase Campylobacter sialyltransferase II (cstII) from C.jejuni serotype O10 (ORF 7a of lipooligosaccharide (LOS) biosynthesislocus) 4 atg aaa aaa gtt att att gct gga aat gga cca agt tta aaa gaa att48 Met Lys Lys Val Ile Ile Ala Gly Asn Gly Pro Ser Leu Lys Glu Ile 1 510 15 gat tat tca agg cta cca aat gat ttt gat gta ttt aga tgc aat caa 96Asp Tyr Ser Arg Leu Pro Asn Asp Phe Asp Val Phe Arg Cys Asn Gln 20 25 30ttt tat ttt gaa gat aaa tac tat ctt ggt aaa aaa ttc aaa gca gta 144 PheTyr Phe Glu Asp Lys Tyr Tyr Leu Gly Lys Lys Phe Lys Ala Val 35 40 45 ttttac aat cct ggt ctt ttt ttt gaa caa tac tac act tta aaa cat 192 Phe TyrAsn Pro Gly Leu Phe Phe Glu Gln Tyr Tyr Thr Leu Lys His 50 55 60 tta atccaa aat caa gaa tat gag acc gaa cta att atg tgt tct aat 240 Leu Ile GlnAsn Gln Glu Tyr Glu Thr Glu Leu Ile Met Cys Ser Asn 65 70 75 80 tac aaccaa gct cat cta gaa aat gaa aat ttt gta aaa act ttt tac 288 Tyr Asn GlnAla His Leu Glu Asn Glu Asn Phe Val Lys Thr Phe Tyr 85 90 95 gat tat tttcct gat gct cat ttg gga tat gat ttt ttt aaa caa ctt 336 Asp Tyr Phe ProAsp Ala His Leu Gly Tyr Asp Phe Phe Lys Gln Leu 100 105 110 aaa gaa tttaat gct tat ttt aaa ttt cac gaa att tat ctc aat caa 384 Lys Glu Phe AsnAla Tyr Phe Lys Phe His Glu Ile Tyr Leu Asn Gln 115 120 125 aga att acctca gga gtc tat atg tgt gca gta gct ata gcc cta gga 432 Arg Ile Thr SerGly Val Tyr Met Cys Ala Val Ala Ile Ala Leu Gly 130 135 140 tac aaa gaaatt tat ctt tct gga att gat ttt tat caa aat ggg tca 480 Tyr Lys Glu IleTyr Leu Ser Gly Ile Asp Phe Tyr Gln Asn Gly Ser 145 150 155 160 tct tatgct ttt gat acc aaa caa gaa aat ctt tta aaa ctg gct cct 528 Ser Tyr AlaPhe Asp Thr Lys Gln Glu Asn Leu Leu Lys Leu Ala Pro 165 170 175 gat tttaaa aat gat cgc tca cac tat atc gga cat agt aaa aat aca 576 Asp Phe LysAsn Asp Arg Ser His Tyr Ile Gly His Ser Lys Asn Thr 180 185 190 gat ataaaa gct tta gaa ttt cta gaa aaa act tac aaa ata aaa cta 624 Asp Ile LysAla Leu Glu Phe Leu Glu Lys Thr Tyr Lys Ile Lys Leu 195 200 205 tat tgctta tgt cct aac agt ctt tta gca aat ttt ata gaa cta gcg 672 Tyr Cys LeuCys Pro Asn Ser Leu Leu Ala Asn Phe Ile Glu Leu Ala 210 215 220 cca aattta aat tca aat ttt atc ata caa gaa aaa aat aac tac act 720 Pro Asn LeuAsn Ser Asn Phe Ile Ile Gln Glu Lys Asn Asn Tyr Thr 225 230 235 240 aaagat ata ctc ata cct tct agt gag gct tat gga aaa ttt tca aaa 768 Lys AspIle Leu Ile Pro Ser Ser Glu Ala Tyr Gly Lys Phe Ser Lys 245 250 255 aatatt aat ttt aaa aaa ata aaa att aaa gaa aat att tat tac aag 816 Asn IleAsn Phe Lys Lys Ile Lys Ile Lys Glu Asn Ile Tyr Tyr Lys 260 265 270 ttgata aaa gat cta tta aga tta cct agt gat ata aag cat tat ttc 864 Leu IleLys Asp Leu Leu Arg Leu Pro Ser Asp Ile Lys His Tyr Phe 275 280 285 aaagga aaa taa 876 Lys Gly Lys 290 5 291 PRT Campylobacter jejunibifunctional alpha-2,3/alpha 2,8-sialyltransferase Campylobactersialyltransferase II (cstII) from C. jejuni serotype O10 (ORF 7a oflipooligosaccharide (LOS) biosynthesis locus) 5 Met Lys Lys Val Ile IleAla Gly Asn Gly Pro Ser Leu Lys Glu Ile 1 5 10 15 Asp Tyr Ser Arg LeuPro Asn Asp Phe Asp Val Phe Arg Cys Asn Gln 20 25 30 Phe Tyr Phe Glu AspLys Tyr Tyr Leu Gly Lys Lys Phe Lys Ala Val 35 40 45 Phe Tyr Asn Pro GlyLeu Phe Phe Glu Gln Tyr Tyr Thr Leu Lys His 50 55 60 Leu Ile Gln Asn GlnGlu Tyr Glu Thr Glu Leu Ile Met Cys Ser Asn 65 70 75 80 Tyr Asn Gln AlaHis Leu Glu Asn Glu Asn Phe Val Lys Thr Phe Tyr 85 90 95 Asp Tyr Phe ProAsp Ala His Leu Gly Tyr Asp Phe Phe Lys Gln Leu 100 105 110 Lys Glu PheAsn Ala Tyr Phe Lys Phe His Glu Ile Tyr Leu Asn Gln 115 120 125 Arg IleThr Ser Gly Val Tyr Met Cys Ala Val Ala Ile Ala Leu Gly 130 135 140 TyrLys Glu Ile Tyr Leu Ser Gly Ile Asp Phe Tyr Gln Asn Gly Ser 145 150 155160 Ser Tyr Ala Phe Asp Thr Lys Gln Glu Asn Leu Leu Lys Leu Ala Pro 165170 175 Asp Phe Lys Asn Asp Arg Ser His Tyr Ile Gly His Ser Lys Asn Thr180 185 190 Asp Ile Lys Ala Leu Glu Phe Leu Glu Lys Thr Tyr Lys Ile LysLeu 195 200 205 Tyr Cys Leu Cys Pro Asn Ser Leu Leu Ala Asn Phe Ile GluLeu Ala 210 215 220 Pro Asn Leu Asn Ser Asn Phe Ile Ile Gln Glu Lys AsnAsn Tyr Thr 225 230 235 240 Lys Asp Ile Leu Ile Pro Ser Ser Glu Ala TyrGly Lys Phe Ser Lys 245 250 255 Asn Ile Asn Phe Lys Lys Ile Lys Ile LysGlu Asn Ile Tyr Tyr Lys 260 265 270 Leu Ile Lys Asp Leu Leu Arg Leu ProSer Asp Ile Lys His Tyr Phe 275 280 285 Lys Gly Lys 290 6 876 DNACampylobacter jejuni CDS (1)..(876) Campylobacter alpha-2,3/alpha2,8-sialyltransferase II (cstII) from C. jejuni serotype O41 6 atg aaaaaa gtt att att gct gga aat gga cca agt tta aaa gaa att 48 Met Lys LysVal Ile Ile Ala Gly Asn Gly Pro Ser Leu Lys Glu Ile 1 5 10 15 gat tattca aga cta cca aat gat ttt gat gta ttt aga tgc aat caa 96 Asp Tyr SerArg Leu Pro Asn Asp Phe Asp Val Phe Arg Cys Asn Gln 20 25 30 ttt tat tttgaa gat aaa tac tat ctt ggt aaa aaa tgc aaa gca gta 144 Phe Tyr Phe GluAsp Lys Tyr Tyr Leu Gly Lys Lys Cys Lys Ala Val 35 40 45 ttt tac aat cctagt ctt ttt ttt gaa caa tac tac act tta aaa cat 192 Phe Tyr Asn Pro SerLeu Phe Phe Glu Gln Tyr Tyr Thr Leu Lys His 50 55 60 tta atc caa aat caagaa tat gag acc gaa cta atc atg tgt tct aat 240 Leu Ile Gln Asn Gln GluTyr Glu Thr Glu Leu Ile Met Cys Ser Asn 65 70 75 80 ttt aac caa gct catcta gaa aat caa aat ttt gta aaa act ttt tac 288 Phe Asn Gln Ala His LeuGlu Asn Gln Asn Phe Val Lys Thr Phe Tyr 85 90 95 gat tat ttt cct gat gctcat ttg gga tat gat ttt ttc aaa caa ctt 336 Asp Tyr Phe Pro Asp Ala HisLeu Gly Tyr Asp Phe Phe Lys Gln Leu 100 105 110 aaa gaa ttc aat gct tatttt aaa ttt cac gaa att tat ttc aat caa 384 Lys Glu Phe Asn Ala Tyr PheLys Phe His Glu Ile Tyr Phe Asn Gln 115 120 125 aga att acc tca ggg gtctat atg tgc aca gta gcc ata gcc cta gga 432 Arg Ile Thr Ser Gly Val TyrMet Cys Thr Val Ala Ile Ala Leu Gly 130 135 140 tac aaa gaa att tat ctttcg gga att gat ttt tat caa aat gga tca 480 Tyr Lys Glu Ile Tyr Leu SerGly Ile Asp Phe Tyr Gln Asn Gly Ser 145 150 155 160 tct tat gct ttt gatacc aaa caa aaa aat ctt tta aaa ttg gct cct 528 Ser Tyr Ala Phe Asp ThrLys Gln Lys Asn Leu Leu Lys Leu Ala Pro 165 170 175 aat ttt aaa aat gataat tca cac tat atc gga cat agt aaa aat aca 576 Asn Phe Lys Asn Asp AsnSer His Tyr Ile Gly His Ser Lys Asn Thr 180 185 190 gat ata aaa gct ttagaa ttt cta gaa aaa act tac gaa ata aag cta 624 Asp Ile Lys Ala Leu GluPhe Leu Glu Lys Thr Tyr Glu Ile Lys Leu 195 200 205 tat tgt tta tgt cctaac agt ctt tta gca aat ttt ata gaa cta gcg 672 Tyr Cys Leu Cys Pro AsnSer Leu Leu Ala Asn Phe Ile Glu Leu Ala 210 215 220 cca aat tta aat tcaaat ttt atc ata caa gaa aaa aat aac tat act 720 Pro Asn Leu Asn Ser AsnPhe Ile Ile Gln Glu Lys Asn Asn Tyr Thr 225 230 235 240 aaa gat ata ctcata cct tct agt gag gct tat gga aaa ttt aca aaa 768 Lys Asp Ile Leu IlePro Ser Ser Glu Ala Tyr Gly Lys Phe Thr Lys 245 250 255 aat att aat tttaaa aaa ata aaa att aaa gaa aat att tat tac aag 816 Asn Ile Asn Phe LysLys Ile Lys Ile Lys Glu Asn Ile Tyr Tyr Lys 260 265 270 ttg ata aaa gatcta tta aga tta cct agt gat ata aag cat tat ttc 864 Leu Ile Lys Asp LeuLeu Arg Leu Pro Ser Asp Ile Lys His Tyr Phe 275 280 285 aaa gga aaa taa876 Lys Gly Lys 290 7 291 PRT Campylobacter jejuni Campylobacteralpha-2,3/alpha 2,8-sialyltransferase II (cstII) from C. jejuni serotypeO41 7 Met Lys Lys Val Ile Ile Ala Gly Asn Gly Pro Ser Leu Lys Glu Ile 15 10 15 Asp Tyr Ser Arg Leu Pro Asn Asp Phe Asp Val Phe Arg Cys Asn Gln20 25 30 Phe Tyr Phe Glu Asp Lys Tyr Tyr Leu Gly Lys Lys Cys Lys Ala Val35 40 45 Phe Tyr Asn Pro Ser Leu Phe Phe Glu Gln Tyr Tyr Thr Leu Lys His50 55 60 Leu Ile Gln Asn Gln Glu Tyr Glu Thr Glu Leu Ile Met Cys Ser Asn65 70 75 80 Phe Asn Gln Ala His Leu Glu Asn Gln Asn Phe Val Lys Thr PheTyr 85 90 95 Asp Tyr Phe Pro Asp Ala His Leu Gly Tyr Asp Phe Phe Lys GlnLeu 100 105 110 Lys Glu Phe Asn Ala Tyr Phe Lys Phe His Glu Ile Tyr PheAsn Gln 115 120 125 Arg Ile Thr Ser Gly Val Tyr Met Cys Thr Val Ala IleAla Leu Gly 130 135 140 Tyr Lys Glu Ile Tyr Leu Ser Gly Ile Asp Phe TyrGln Asn Gly Ser 145 150 155 160 Ser Tyr Ala Phe Asp Thr Lys Gln Lys AsnLeu Leu Lys Leu Ala Pro 165 170 175 Asn Phe Lys Asn Asp Asn Ser His TyrIle Gly His Ser Lys Asn Thr 180 185 190 Asp Ile Lys Ala Leu Glu Phe LeuGlu Lys Thr Tyr Glu Ile Lys Leu 195 200 205 Tyr Cys Leu Cys Pro Asn SerLeu Leu Ala Asn Phe Ile Glu Leu Ala 210 215 220 Pro Asn Leu Asn Ser AsnPhe Ile Ile Gln Glu Lys Asn Asn Tyr Thr 225 230 235 240 Lys Asp Ile LeuIle Pro Ser Ser Glu Ala Tyr Gly Lys Phe Thr Lys 245 250 255 Asn Ile AsnPhe Lys Lys Ile Lys Ile Lys Glu Asn Ile Tyr Tyr Lys 260 265 270 Leu IleLys Asp Leu Leu Arg Leu Pro Ser Asp Ile Lys His Tyr Phe 275 280 285 LysGly Lys 290 8 876 DNA Campylobacter jejuni CDS (1)..(876) Campylobacteralpha-2,3/alpha 2,8-sialyltransferase II (CstII) from C. jejuni O19 8atg aaa aaa gtt att att gct gga aat gga cca agt tta aaa gaa att 48 MetLys Lys Val Ile Ile Ala Gly Asn Gly Pro Ser Leu Lys Glu Ile 1 5 10 15gat tat tca agg cta cca aat gat ttt gat gta ttt aga tgt aat caa 96 AspTyr Ser Arg Leu Pro Asn Asp Phe Asp Val Phe Arg Cys Asn Gln 20 25 30 ttttat ttt gaa gat aaa tac tat ctt ggt aaa aaa tgc aaa gca gtg 144 Phe TyrPhe Glu Asp Lys Tyr Tyr Leu Gly Lys Lys Cys Lys Ala Val 35 40 45 ttt tacacc cct aat ttc ttc ttt gag caa tac tac act tta aaa cat 192 Phe Tyr ThrPro Asn Phe Phe Phe Glu Gln Tyr Tyr Thr Leu Lys His 50 55 60 tta atc caaaat caa gaa tat gag acc gaa cta att atg tgt tct aat 240 Leu Ile Gln AsnGln Glu Tyr Glu Thr Glu Leu Ile Met Cys Ser Asn 65 70 75 80 tac aac caagct cat cta gaa aat gaa aat ttt gta aaa act ttt tac 288 Tyr Asn Gln AlaHis Leu Glu Asn Glu Asn Phe Val Lys Thr Phe Tyr 85 90 95 gat tat ttt cctgat gct cat ttg gga tat gat ttt ttt aaa caa ctt 336 Asp Tyr Phe Pro AspAla His Leu Gly Tyr Asp Phe Phe Lys Gln Leu 100 105 110 aaa gaa ttt aatgct tat ttt aaa ttt cac gaa att tat ttc aat caa 384 Lys Glu Phe Asn AlaTyr Phe Lys Phe His Glu Ile Tyr Phe Asn Gln 115 120 125 aga att acc tcaggg gtc tat atg tgt gca gta gcc ata gcc cta gga 432 Arg Ile Thr Ser GlyVal Tyr Met Cys Ala Val Ala Ile Ala Leu Gly 130 135 140 tac aaa gaa atttat ctt tcg gga att gat ttt tat caa aat ggg tca 480 Tyr Lys Glu Ile TyrLeu Ser Gly Ile Asp Phe Tyr Gln Asn Gly Ser 145 150 155 160 tct tat gctttt gat acc aaa caa gaa aat ctt tta aaa cta gcc cct 528 Ser Tyr Ala PheAsp Thr Lys Gln Glu Asn Leu Leu Lys Leu Ala Pro 165 170 175 gat ttt aaaaat gat cgc tcg cac tat atc gga cat agt aaa aat aca 576 Asp Phe Lys AsnAsp Arg Ser His Tyr Ile Gly His Ser Lys Asn Thr 180 185 190 gat ata aaagct tta gaa ttt cta gaa aaa act tac aaa ata aaa cta 624 Asp Ile Lys AlaLeu Glu Phe Leu Glu Lys Thr Tyr Lys Ile Lys Leu 195 200 205 tat tgc ttatgt cct aat agt ctt tta gca aat ttt ata gaa cta gcg 672 Tyr Cys Leu CysPro Asn Ser Leu Leu Ala Asn Phe Ile Glu Leu Ala 210 215 220 cca aat ttaaat tca aat ttt atc ata caa gaa aaa aat aac tac act 720 Pro Asn Leu AsnSer Asn Phe Ile Ile Gln Glu Lys Asn Asn Tyr Thr 225 230 235 240 aaa gatata ctc ata cct tct agt gag gct tat gga aaa ttt tca aaa 768 Lys Asp IleLeu Ile Pro Ser Ser Glu Ala Tyr Gly Lys Phe Ser Lys 245 250 255 aat attaat ttt aaa aaa ata aaa att aaa gaa aat gtt tat tac aag 816 Asn Ile AsnPhe Lys Lys Ile Lys Ile Lys Glu Asn Val Tyr Tyr Lys 260 265 270 ttg ataaaa gat cta tta aga tta cct agt gat ata aag cat tat ttc 864 Leu Ile LysAsp Leu Leu Arg Leu Pro Ser Asp Ile Lys His Tyr Phe 275 280 285 aaa ggaaaa taa 876 Lys Gly Lys 290 9 291 PRT Campylobacter jejuni Campylobacteralpha-2,3/alpha 2,8-sialyltransferase II (CstII) from C. jejuni O19 9Met Lys Lys Val Ile Ile Ala Gly Asn Gly Pro Ser Leu Lys Glu Ile 1 5 1015 Asp Tyr Ser Arg Leu Pro Asn Asp Phe Asp Val Phe Arg Cys Asn Gln 20 2530 Phe Tyr Phe Glu Asp Lys Tyr Tyr Leu Gly Lys Lys Cys Lys Ala Val 35 4045 Phe Tyr Thr Pro Asn Phe Phe Phe Glu Gln Tyr Tyr Thr Leu Lys His 50 5560 Leu Ile Gln Asn Gln Glu Tyr Glu Thr Glu Leu Ile Met Cys Ser Asn 65 7075 80 Tyr Asn Gln Ala His Leu Glu Asn Glu Asn Phe Val Lys Thr Phe Tyr 8590 95 Asp Tyr Phe Pro Asp Ala His Leu Gly Tyr Asp Phe Phe Lys Gln Leu100 105 110 Lys Glu Phe Asn Ala Tyr Phe Lys Phe His Glu Ile Tyr Phe AsnGln 115 120 125 Arg Ile Thr Ser Gly Val Tyr Met Cys Ala Val Ala Ile AlaLeu Gly 130 135 140 Tyr Lys Glu Ile Tyr Leu Ser Gly Ile Asp Phe Tyr GlnAsn Gly Ser 145 150 155 160 Ser Tyr Ala Phe Asp Thr Lys Gln Glu Asn LeuLeu Lys Leu Ala Pro 165 170 175 Asp Phe Lys Asn Asp Arg Ser His Tyr IleGly His Ser Lys Asn Thr 180 185 190 Asp Ile Lys Ala Leu Glu Phe Leu GluLys Thr Tyr Lys Ile Lys Leu 195 200 205 Tyr Cys Leu Cys Pro Asn Ser LeuLeu Ala Asn Phe Ile Glu Leu Ala 210 215 220 Pro Asn Leu Asn Ser Asn PheIle Ile Gln Glu Lys Asn Asn Tyr Thr 225 230 235 240 Lys Asp Ile Leu IlePro Ser Ser Glu Ala Tyr Gly Lys Phe Ser Lys 245 250 255 Asn Ile Asn PheLys Lys Ile Lys Ile Lys Glu Asn Val Tyr Tyr Lys 260 265 270 Leu Ile LysAsp Leu Leu Arg Leu Pro Ser Asp Ile Lys His Tyr Phe 275 280 285 Lys GlyLys 290 10 294 PRT Campylobacter jejuni Campylobacter alpha-2,3/alpha2,8-sialyltransferase II (CstII) from C. jejuni strain NCTC 11168 10 MetSer Met Asn Ile Asn Ala Leu Val Cys Gly Asn Gly Pro Ser Leu 1 5 10 15Lys Asn Ile Asp Tyr Lys Arg Leu Pro Lys Gln Phe Asp Val Phe Arg 20 25 30Cys Asn Gln Phe Tyr Phe Glu Asp Arg Tyr Phe Val Gly Lys Asp Val 35 40 45Lys Tyr Val Phe Phe Asn Pro Phe Val Phe Phe Glu Gln Tyr Tyr Thr 50 55 60Ser Lys Lys Leu Ile Gln Asn Glu Glu Tyr Asn Ile Glu Asn Ile Val 65 70 7580 Cys Ser Thr Ile Asn Leu Glu Tyr Ile Asp Gly Phe Gln Phe Val Asp 85 9095 Asn Phe Glu Leu Tyr Phe Ser Asp Ala Phe Leu Gly His Glu Ile Ile 100105 110 Lys Lys Leu Lys Asp Phe Phe Ala Tyr Ile Lys Tyr Asn Glu Ile Tyr115 120 125 Asn Arg Gln Arg Ile Thr Ser Gly Val Tyr Met Cys Ala Thr AlaVal 130 135 140 Ala Leu Gly Tyr Lys Ser Ile Tyr Ile Ser Gly Ile Asp PheTyr Gln 145 150 155 160 Asp Thr Asn Asn Leu Tyr Ala Phe Asp Asn Asn LysLys Asn Leu Leu 165 170 175 Asn Lys Cys Thr Gly Phe Lys Asn Gln Lys PheLys Phe Ile Asn His 180 185 190 Ser Met Ala Cys Asp Leu Gln Ala Leu AspTyr Leu Met Lys Arg Tyr 195 200 205 Asp Val Asn Ile Tyr Ser Leu Asn SerAsp Glu Tyr Phe Lys Leu Ala 210 215 220 Pro Asp Ile Gly Ser Asp Phe ValLeu Ser Lys Lys Pro Lys Lys Tyr 225 230 235 240 Ile Asn Asp Ile Leu IlePro Asp Lys Tyr Ala Gln Glu Arg Tyr Tyr 245 250 255 Gly Lys Lys Ser ArgLeu Lys Glu Asn Leu His Tyr Lys Leu Ile Lys 260 265 270 Asp Leu Ile ArgLeu Pro Ser Asp Ile Lys His Tyr Leu Lys Glu Lys 275 280 285 Tyr Ala AsnLys Asn Arg 290 11 873 DNA Campylobacter jejuni CDS (1)..(873)Campylobacter alpha-2,3/alpha 2,8-sialyltransferase II (CstII) from C.jejuni O4 11 atg aaa aaa gtt att att gct gga aat gga cca agt tta aaa gaaatt 48 Met Lys Lys Val Ile Ile Ala Gly Asn Gly Pro Ser Leu Lys Glu Ile 15 10 15 gat tat tca agg cta cca aat gat ttt gat gta ttt aga tgt aat caa96 Asp Tyr Ser Arg Leu Pro Asn Asp Phe Asp Val Phe Arg Cys Asn Gln 20 2530 ttt tat ttt gaa gat aaa tac tat ctt ggt aaa aaa tgc aaa gca gtg 144Phe Tyr Phe Glu Asp Lys Tyr Tyr Leu Gly Lys Lys Cys Lys Ala Val 35 40 45ttt tac acc cct ggt ttc ttc ttt gag caa tac tac act tta aaa cat 192 PheTyr Thr Pro Gly Phe Phe Phe Glu Gln Tyr Tyr Thr Leu Lys His 50 55 60 ttaatc caa aat caa gaa tat gag acc gaa cta att atg tgt tct aat 240 Leu IleGln Asn Gln Glu Tyr Glu Thr Glu Leu Ile Met Cys Ser Asn 65 70 75 80 tacaac caa gct cat cta gaa aat gaa aat ttt gta aaa act ttt tac 288 Tyr AsnGln Ala His Leu Glu Asn Glu Asn Phe Val Lys Thr Phe Tyr 85 90 95 gat tatttt cct gat gct cat ttg gga tat gat ttt ttt aaa caa ctt 336 Asp Tyr PhePro Asp Ala His Leu Gly Tyr Asp Phe Phe Lys Gln Leu 100 105 110 aaa gaattt aat gct tat ttt aaa ttt cac gaa att tat ttc aat caa 384 Lys Glu PheAsn Ala Tyr Phe Lys Phe His Glu Ile Tyr Phe Asn Gln 115 120 125 aga attacc tca ggg gtc tat atg tgt gca gta gcc ata gcc cta gga 432 Arg Ile ThrSer Gly Val Tyr Met Cys Ala Val Ala Ile Ala Leu Gly 130 135 140 tac aaagaa att tat ctt tcg gga att gat ttt tat caa aat ggg tca 480 Tyr Lys GluIle Tyr Leu Ser Gly Ile Asp Phe Tyr Gln Asn Gly Ser 145 150 155 160 tcttat gct ttt gat acc aaa caa gaa aat ctt tta aaa cta gcc cct 528 Ser TyrAla Phe Asp Thr Lys Gln Glu Asn Leu Leu Lys Leu Ala Pro 165 170 175 gatttt aaa aat gat cgc tca cac tat atc gga cat agt aaa aat aca 576 Asp PheLys Asn Asp Arg Ser His Tyr Ile Gly His Ser Lys Asn Thr 180 185 190 gatata aaa gct tta gaa ttt cta gaa aaa act tac aaa ata aaa cta 624 Asp IleLys Ala Leu Glu Phe Leu Glu Lys Thr Tyr Lys Ile Lys Leu 195 200 205 tattgc tta tgt cct aac agt ctt tta gca aat ttt ata gaa cta gcg 672 Tyr CysLeu Cys Pro Asn Ser Leu Leu Ala Asn Phe Ile Glu Leu Ala 210 215 220 ccaaat tta aat tca aat ttt atc ata caa gaa aaa aat aac tac act 720 Pro AsnLeu Asn Ser Asn Phe Ile Ile Gln Glu Lys Asn Asn Tyr Thr 225 230 235 240aaa gat ata ctc ata cct tct agt gag gct tat gga aaa ttt tca aaa 768 LysAsp Ile Leu Ile Pro Ser Ser Glu Ala Tyr Gly Lys Phe Ser Lys 245 250 255aat att aat ttt aaa aaa ata aaa att aaa gaa aat gtt tat tac aag 816 AsnIle Asn Phe Lys Lys Ile Lys Ile Lys Glu Asn Val Tyr Tyr Lys 260 265 270ttg ata aaa gat cta tta aga tta cct agt gat ata aag cat tat ttc 864 LeuIle Lys Asp Leu Leu Arg Leu Pro Ser Asp Ile Lys His Tyr Phe 275 280 285aaa gga aaa 873 Lys Gly Lys 290 12 291 PRT Campylobacter jejuniCampylobacter alpha-2,3/alpha 2,8-sialyltransferase II (CstII) from C.jejuni O4 12 Met Lys Lys Val Ile Ile Ala Gly Asn Gly Pro Ser Leu Lys GluIle 1 5 10 15 Asp Tyr Ser Arg Leu Pro Asn Asp Phe Asp Val Phe Arg CysAsn Gln 20 25 30 Phe Tyr Phe Glu Asp Lys Tyr Tyr Leu Gly Lys Lys Cys LysAla Val 35 40 45 Phe Tyr Thr Pro Gly Phe Phe Phe Glu Gln Tyr Tyr Thr LeuLys His 50 55 60 Leu Ile Gln Asn Gln Glu Tyr Glu Thr Glu Leu Ile Met CysSer Asn 65 70 75 80 Tyr Asn Gln Ala His Leu Glu Asn Glu Asn Phe Val LysThr Phe Tyr 85 90 95 Asp Tyr Phe Pro Asp Ala His Leu Gly Tyr Asp Phe PheLys Gln Leu 100 105 110 Lys Glu Phe Asn Ala Tyr Phe Lys Phe His Glu IleTyr Phe Asn Gln 115 120 125 Arg Ile Thr Ser Gly Val Tyr Met Cys Ala ValAla Ile Ala Leu Gly 130 135 140 Tyr Lys Glu Ile Tyr Leu Ser Gly Ile AspPhe Tyr Gln Asn Gly Ser 145 150 155 160 Ser Tyr Ala Phe Asp Thr Lys GlnGlu Asn Leu Leu Lys Leu Ala Pro 165 170 175 Asp Phe Lys Asn Asp Arg SerHis Tyr Ile Gly His Ser Lys Asn Thr 180 185 190 Asp Ile Lys Ala Leu GluPhe Leu Glu Lys Thr Tyr Lys Ile Lys Leu 195 200 205 Tyr Cys Leu Cys ProAsn Ser Leu Leu Ala Asn Phe Ile Glu Leu Ala 210 215 220 Pro Asn Leu AsnSer Asn Phe Ile Ile Gln Glu Lys Asn Asn Tyr Thr 225 230 235 240 Lys AspIle Leu Ile Pro Ser Ser Glu Ala Tyr Gly Lys Phe Ser Lys 245 250 255 AsnIle Asn Phe Lys Lys Ile Lys Ile Lys Glu Asn Val Tyr Tyr Lys 260 265 270Leu Ile Lys Asp Leu Leu Arg Leu Pro Ser Asp Ile Lys His Tyr Phe 275 280285 Lys Gly Lys 290 13 873 DNA Campylobacter jejuni CDS (1)..(873)Campylobacter alpha-2,3/alpha 2,8-sialyltransferase II (CstII) from C.jejuni O36 13 atg aaa aaa gtt att att gct gga aat gga cca agt tta aaagaa att 48 Met Lys Lys Val Ile Ile Ala Gly Asn Gly Pro Ser Leu Lys GluIle 1 5 10 15 gat tat tca agg cta cca aat gat ttt gat gta ttt aga tgtaat caa 96 Asp Tyr Ser Arg Leu Pro Asn Asp Phe Asp Val Phe Arg Cys AsnGln 20 25 30 ttt tat ttt gaa gat aaa tac tat ctt ggt aaa aaa tgc aaa acagtg 144 Phe Tyr Phe Glu Asp Lys Tyr Tyr Leu Gly Lys Lys Cys Lys Thr Val35 40 45 ttt tac acc cct aat ttc ttc ttt gag caa tac tac act tta aaa cat192 Phe Tyr Thr Pro Asn Phe Phe Phe Glu Gln Tyr Tyr Thr Leu Lys His 5055 60 tta atc caa aat caa gaa tat gag acc gaa cta att atg tgt tct aat240 Leu Ile Gln Asn Gln Glu Tyr Glu Thr Glu Leu Ile Met Cys Ser Asn 6570 75 80 tac aac caa gct cat cta gaa aat gaa aat ttt gta aaa act ttt tac288 Tyr Asn Gln Ala His Leu Glu Asn Glu Asn Phe Val Lys Thr Phe Tyr 8590 95 gat tat ttt cct gat gct cat ttg gga tat gat ttt ttt aaa caa ctt336 Asp Tyr Phe Pro Asp Ala His Leu Gly Tyr Asp Phe Phe Lys Gln Leu 100105 110 aaa gaa ttt aat gct tat ttt aaa ttt cac gaa att tat ttc aat caa384 Lys Glu Phe Asn Ala Tyr Phe Lys Phe His Glu Ile Tyr Phe Asn Gln 115120 125 aga att acc tca ggg gtc tat atg tgt gca gta gcc ata gcc cta gga432 Arg Ile Thr Ser Gly Val Tyr Met Cys Ala Val Ala Ile Ala Leu Gly 130135 140 tac aaa gaa att tat ctt tcg gga att gat ttt tat caa aat ggg tca480 Tyr Lys Glu Ile Tyr Leu Ser Gly Ile Asp Phe Tyr Gln Asn Gly Ser 145150 155 160 tct tat gct ttt gat acc aaa caa gaa aat ctt tta aaa cta gcccct 528 Ser Tyr Ala Phe Asp Thr Lys Gln Glu Asn Leu Leu Lys Leu Ala Pro165 170 175 gat ttt aaa aat gat cgc tca cac tat atc gga cat agt aaa aataca 576 Asp Phe Lys Asn Asp Arg Ser His Tyr Ile Gly His Ser Lys Asn Thr180 185 190 gat ata aaa gct tta gaa ttt cta gaa aaa act tac aaa ata aaacta 624 Asp Ile Lys Ala Leu Glu Phe Leu Glu Lys Thr Tyr Lys Ile Lys Leu195 200 205 tat tgc tta tgt cct aat agt ctt tta gca aat ttt ata gaa ctagcg 672 Tyr Cys Leu Cys Pro Asn Ser Leu Leu Ala Asn Phe Ile Glu Leu Ala210 215 220 cca aat tta aat tca aat ttt atc ata caa gaa aaa aat aac tacact 720 Pro Asn Leu Asn Ser Asn Phe Ile Ile Gln Glu Lys Asn Asn Tyr Thr225 230 235 240 aaa gat ata ctc ata cct tct agt gag gct tat gga aaa ttttca aaa 768 Lys Asp Ile Leu Ile Pro Ser Ser Glu Ala Tyr Gly Lys Phe SerLys 245 250 255 aat att aat ttt aaa aaa ata aaa att aaa gaa aat gtt tattac aag 816 Asn Ile Asn Phe Lys Lys Ile Lys Ile Lys Glu Asn Val Tyr TyrLys 260 265 270 ttg ata aaa gat cta tta aga tta cct agt gat ata aag cattat ttc 864 Leu Ile Lys Asp Leu Leu Arg Leu Pro Ser Asp Ile Lys His TyrPhe 275 280 285 aaa gga aaa 873 Lys Gly Lys 290 14 291 PRT Campylobacterjejuni Campylobacter alpha-2,3/alpha 2,8-sialyltransferase II (CstII)from C. jejuni O36 14 Met Lys Lys Val Ile Ile Ala Gly Asn Gly Pro SerLeu Lys Glu Ile 1 5 10 15 Asp Tyr Ser Arg Leu Pro Asn Asp Phe Asp ValPhe Arg Cys Asn Gln 20 25 30 Phe Tyr Phe Glu Asp Lys Tyr Tyr Leu Gly LysLys Cys Lys Thr Val 35 40 45 Phe Tyr Thr Pro Asn Phe Phe Phe Glu Gln TyrTyr Thr Leu Lys His 50 55 60 Leu Ile Gln Asn Gln Glu Tyr Glu Thr Glu LeuIle Met Cys Ser Asn 65 70 75 80 Tyr Asn Gln Ala His Leu Glu Asn Glu AsnPhe Val Lys Thr Phe Tyr 85 90 95 Asp Tyr Phe Pro Asp Ala His Leu Gly TyrAsp Phe Phe Lys Gln Leu 100 105 110 Lys Glu Phe Asn Ala Tyr Phe Lys PheHis Glu Ile Tyr Phe Asn Gln 115 120 125 Arg Ile Thr Ser Gly Val Tyr MetCys Ala Val Ala Ile Ala Leu Gly 130 135 140 Tyr Lys Glu Ile Tyr Leu SerGly Ile Asp Phe Tyr Gln Asn Gly Ser 145 150 155 160 Ser Tyr Ala Phe AspThr Lys Gln Glu Asn Leu Leu Lys Leu Ala Pro 165 170 175 Asp Phe Lys AsnAsp Arg Ser His Tyr Ile Gly His Ser Lys Asn Thr 180 185 190 Asp Ile LysAla Leu Glu Phe Leu Glu Lys Thr Tyr Lys Ile Lys Leu 195 200 205 Tyr CysLeu Cys Pro Asn Ser Leu Leu Ala Asn Phe Ile Glu Leu Ala 210 215 220 ProAsn Leu Asn Ser Asn Phe Ile Ile Gln Glu Lys Asn Asn Tyr Thr 225 230 235240 Lys Asp Ile Leu Ile Pro Ser Ser Glu Ala Tyr Gly Lys Phe Ser Lys 245250 255 Asn Ile Asn Phe Lys Lys Ile Lys Ile Lys Glu Asn Val Tyr Tyr Lys260 265 270 Leu Ile Lys Asp Leu Leu Arg Leu Pro Ser Asp Ile Lys His TyrPhe 275 280 285 Lys Gly Lys 290 15 1170 DNA Campylobacter jejuniglycosyltransferase from C. jejuni strain OH4384 (ORF 4a oflipooligosaccharide (LOS) biosynthesis locus) 15 atgaagaaaa taggtgtagttataccaatc tataatgtag aaaaatattt aagagaatgt 60 ttagatagcg ttatcaatcaaacttatact aacttagaaa tcatacttgt caatgatggt 120 agcacagatg aacactcactcaatattgca aaagaatata ccttaaaaga taaaagaata 180 actctttttg ataagaaaaatgggggttta agttcagcta gaaatatagg tatagaatac 240 tttagcgggg aatataaattaaaaaacaaa actcaacata taaaagaaaa ttctttaata 300 gaatttcaat tggatggtaataatccttat aatatatata aagcatataa aagctctcaa 360 gcttttaata atgaaaaagatttaaccaat tttacttacc ctagtataga ttatattata 420 ttcttagata gtgataattattggaaacta aactgcatag aagaatgcgt tataagaatg 480 aaaaatgtgg atgtattgtggtttgaccat gattgcacct atgaagacaa tataaaaaat 540 aagcacaaaa aaacaaggatggaaattttt gattttaaaa aagaatgtat aatcactcca 600 aaagaatatg caaatcgagcattaagtgta ggatctagag atatttcttt tggatggaat 660 ggaatgattg attttaattttttaaagcaa attaaactta aatttataaa ttttattatc 720 aatgaagata tacactttgggataattttg tttgctagtg ctaataaaat ttatgtttta 780 tcacaaaagt tgtatttgtgtcgtttaaga gcaaacagta tatcaaatca tgataagaag 840 attacaaaag caaatgtgtcagagtatttt aaagatatat atgaaacttt cggggaaaac 900 gctaaggaag caaaaaattatttaaaagca gcaagcaggg ttataactgc tttaaaattg 960 atagaatttt ttaaagatcaaaaaaacgaa aatgcacttg ctataaaaga aacattttta 1020 ccttgctatg ccaaaaaagctttaatgatt aaaaaattta aaaaagatcc tttaaattta 1080 aaggaacaat tagttttaattaaacctttt attcaaacaa aacttcctta tgatatttgg 1140 aaattttggc aaaaaataaaaaatatttaa 1170 16 1044 DNA Campylobacter jejuni CDS (1)..(1044)beta-1,4 N-acetylgalactosaminyl (GalNAc) transferase from C. jejunistrain OH4384 (ORF 5a of lipooligosaccharide (LOS) biosynthesis locus)16 atg cta ttt caa tca tac ttt gtg aaa ata att tgc tta ttc atc cct 48Met Leu Phe Gln Ser Tyr Phe Val Lys Ile Ile Cys Leu Phe Ile Pro 1 5 1015 ttt aga aaa att aga cat aaa ata aaa aaa aca ttt tta cta aaa aac 96Phe Arg Lys Ile Arg His Lys Ile Lys Lys Thr Phe Leu Leu Lys Asn 20 25 30ata caa cga gat aaa atc gat tct tat tta cca aaa aaa act ctt gtg 144 IleGln Arg Asp Lys Ile Asp Ser Tyr Leu Pro Lys Lys Thr Leu Val 35 40 45 caaatt aat aaa tac aac aat gaa gat tta att aaa ctt aat aaa gct 192 Gln IleAsn Lys Tyr Asn Asn Glu Asp Leu Ile Lys Leu Asn Lys Ala 50 55 60 att ataggg gag ggg cat aaa gga tat ttt aat tat gat gaa aaa tct 240 Ile Ile GlyGlu Gly His Lys Gly Tyr Phe Asn Tyr Asp Glu Lys Ser 65 70 75 80 aaa gatcca aaa tct cct ttg aat cct tgg gct ttt ata cga gta aaa 288 Lys Asp ProLys Ser Pro Leu Asn Pro Trp Ala Phe Ile Arg Val Lys 85 90 95 aat gaa gctatt acc tta aaa gct tct ctt gaa agc ata ttg cct gct 336 Asn Glu Ala IleThr Leu Lys Ala Ser Leu Glu Ser Ile Leu Pro Ala 100 105 110 atc caa agaggt gtt ata gga tat aat gat tgt acc gat gga agt gaa 384 Ile Gln Arg GlyVal Ile Gly Tyr Asn Asp Cys Thr Asp Gly Ser Glu 115 120 125 gaa ata attcta gaa ttt tgc aaa caa tat cct tca ttt ata cca ata 432 Glu Ile Ile LeuGlu Phe Cys Lys Gln Tyr Pro Ser Phe Ile Pro Ile 130 135 140 aaa tat ccttat gaa att caa att caa aac cca aaa tca gaa gaa aat 480 Lys Tyr Pro TyrGlu Ile Gln Ile Gln Asn Pro Lys Ser Glu Glu Asn 145 150 155 160 aaa ctctat agc tat tat aat tat gtt gca agt ttt ata cca aaa gat 528 Lys Leu TyrSer Tyr Tyr Asn Tyr Val Ala Ser Phe Ile Pro Lys Asp 165 170 175 gag tggctt ata aaa ata gat gtg gat cat atc tat gat gct aaa aaa 576 Glu Trp LeuIle Lys Ile Asp Val Asp His Ile Tyr Asp Ala Lys Lys 180 185 190 ctt tataaa agc ttc tat ata cca aaa aac aaa tat gat gta gtt agt 624 Leu Tyr LysSer Phe Tyr Ile Pro Lys Asn Lys Tyr Asp Val Val Ser 195 200 205 tat tcaagg gtt gat att cac tat ttt aat gat aat ttt ttt ctt tgt 672 Tyr Ser ArgVal Asp Ile His Tyr Phe Asn Asp Asn Phe Phe Leu Cys 210 215 220 aaa gataat aat ggc aat ata ttg aaa gaa cca gga gat tgc ttg ctt 720 Lys Asp AsnAsn Gly Asn Ile Leu Lys Glu Pro Gly Asp Cys Leu Leu 225 230 235 240 atcaat aat tat aac tta aaa tgg aaa gaa gta tta att gac aga atc 768 Ile AsnAsn Tyr Asn Leu Lys Trp Lys Glu Val Leu Ile Asp Arg Ile 245 250 255 aataac aat tgg aaa aaa gca aca aaa caa agt ttt tct tca aat ata 816 Asn AsnAsn Trp Lys Lys Ala Thr Lys Gln Ser Phe Ser Ser Asn Ile 260 265 270 cactct tta gag caa tta aag tat aaa cac agg ata tta ttt cac act 864 His SerLeu Glu Gln Leu Lys Tyr Lys His Arg Ile Leu Phe His Thr 275 280 285 gaatta aat aat tat cat ttt cct ttt tta aaa aaa cat aga gct caa 912 Glu LeuAsn Asn Tyr His Phe Pro Phe Leu Lys Lys His Arg Ala Gln 290 295 300 gatatt tat aaa tat aat tgg ata agt att gaa gaa ttt aaa aaa ttc 960 Asp IleTyr Lys Tyr Asn Trp Ile Ser Ile Glu Glu Phe Lys Lys Phe 305 310 315 320tat tta caa aat att aat cat aaa ata gaa cct tct atg att tca aaa 1008 TyrLeu Gln Asn Ile Asn His Lys Ile Glu Pro Ser Met Ile Ser Lys 325 330 335gaa act cta aaa aaa ata ttc tta aca ttg ttt taa 1044 Glu Thr Leu Lys LysIle Phe Leu Thr Leu Phe 340 345 17 347 PRT Campylobacter jejuni beta-1,4N-acetylgalactosaminyl (GalNAc) transferase from C. jejuni strain OH4384(ORF 5a of lipooligosaccharide (LOS) biosynthesis locus) 17 Met Leu PheGln Ser Tyr Phe Val Lys Ile Ile Cys Leu Phe Ile Pro 1 5 10 15 Phe ArgLys Ile Arg His Lys Ile Lys Lys Thr Phe Leu Leu Lys Asn 20 25 30 Ile GlnArg Asp Lys Ile Asp Ser Tyr Leu Pro Lys Lys Thr Leu Val 35 40 45 Gln IleAsn Lys Tyr Asn Asn Glu Asp Leu Ile Lys Leu Asn Lys Ala 50 55 60 Ile IleGly Glu Gly His Lys Gly Tyr Phe Asn Tyr Asp Glu Lys Ser 65 70 75 80 LysAsp Pro Lys Ser Pro Leu Asn Pro Trp Ala Phe Ile Arg Val Lys 85 90 95 AsnGlu Ala Ile Thr Leu Lys Ala Ser Leu Glu Ser Ile Leu Pro Ala 100 105 110Ile Gln Arg Gly Val Ile Gly Tyr Asn Asp Cys Thr Asp Gly Ser Glu 115 120125 Glu Ile Ile Leu Glu Phe Cys Lys Gln Tyr Pro Ser Phe Ile Pro Ile 130135 140 Lys Tyr Pro Tyr Glu Ile Gln Ile Gln Asn Pro Lys Ser Glu Glu Asn145 150 155 160 Lys Leu Tyr Ser Tyr Tyr Asn Tyr Val Ala Ser Phe Ile ProLys Asp 165 170 175 Glu Trp Leu Ile Lys Ile Asp Val Asp His Ile Tyr AspAla Lys Lys 180 185 190 Leu Tyr Lys Ser Phe Tyr Ile Pro Lys Asn Lys TyrAsp Val Val Ser 195 200 205 Tyr Ser Arg Val Asp Ile His Tyr Phe Asn AspAsn Phe Phe Leu Cys 210 215 220 Lys Asp Asn Asn Gly Asn Ile Leu Lys GluPro Gly Asp Cys Leu Leu 225 230 235 240 Ile Asn Asn Tyr Asn Leu Lys TrpLys Glu Val Leu Ile Asp Arg Ile 245 250 255 Asn Asn Asn Trp Lys Lys AlaThr Lys Gln Ser Phe Ser Ser Asn Ile 260 265 270 His Ser Leu Glu Gln LeuLys Tyr Lys His Arg Ile Leu Phe His Thr 275 280 285 Glu Leu Asn Asn TyrHis Phe Pro Phe Leu Lys Lys His Arg Ala Gln 290 295 300 Asp Ile Tyr LysTyr Asn Trp Ile Ser Ile Glu Glu Phe Lys Lys Phe 305 310 315 320 Tyr LeuGln Asn Ile Asn His Lys Ile Glu Pro Ser Met Ile Ser Lys 325 330 335 GluThr Leu Lys Lys Ile Phe Leu Thr Leu Phe 340 345 18 1608 DNACampylobacter jejuni CDS (1)..(1608) beta-1,4 N-acetylgalactosaminyl(GalNAc) transferase from C. jejuni O1 18 atg act ttg ttt tat aaa attata gct ttt tta aga ttg ctt aaa att 48 Met Thr Leu Phe Tyr Lys Ile IleAla Phe Leu Arg Leu Leu Lys Ile 1 5 10 15 gat aaa aaa tta aaa ttt gataat gaa tat ttt tta aac tta aat aaa 96 Asp Lys Lys Leu Lys Phe Asp AsnGlu Tyr Phe Leu Asn Leu Asn Lys 20 25 30 aaa atc tac aat gaa aag cat aaaggt ttt ttt gat ttt gat cca aac 144 Lys Ile Tyr Asn Glu Lys His Lys GlyPhe Phe Asp Phe Asp Pro Asn 35 40 45 tca aaa gat aca aaa tct cct tta aatcca tgg gct ttt ata aga gta 192 Ser Lys Asp Thr Lys Ser Pro Leu Asn ProTrp Ala Phe Ile Arg Val 50 55 60 aaa aat gaa gcc act act tta aga gta tcactt gaa agt atg tta cct 240 Lys Asn Glu Ala Thr Thr Leu Arg Val Ser LeuGlu Ser Met Leu Pro 65 70 75 80 gcc ata caa aga ggt gtt ata gga tat aatgat tgt act gat gga agt 288 Ala Ile Gln Arg Gly Val Ile Gly Tyr Asn AspCys Thr Asp Gly Ser 85 90 95 gaa gaa att att ttg gaa ttt tgc aaa caa taccct tcg ttt ata cca 336 Glu Glu Ile Ile Leu Glu Phe Cys Lys Gln Tyr ProSer Phe Ile Pro 100 105 110 gta aaa tat ccc cat gag gtg caa att gaa aatccg caa agc gaa gaa 384 Val Lys Tyr Pro His Glu Val Gln Ile Glu Asn ProGln Ser Glu Glu 115 120 125 aat aaa ctt cat agt tat tat aac tat gta gctagt ttt ata ccg caa 432 Asn Lys Leu His Ser Tyr Tyr Asn Tyr Val Ala SerPhe Ile Pro Gln 130 135 140 gat gag tgg ctt ata aaa ata gat gtg gat cattac tat gat gca aaa 480 Asp Glu Trp Leu Ile Lys Ile Asp Val Asp His TyrTyr Asp Ala Lys 145 150 155 160 aaa tta tat aag agt ttt tat atg gca tcaaaa aat act gct gtt aga 528 Lys Leu Tyr Lys Ser Phe Tyr Met Ala Ser LysAsn Thr Ala Val Arg 165 170 175 ttt cca aga att aat ttt tta ata cta gataaa att gta att caa aat 576 Phe Pro Arg Ile Asn Phe Leu Ile Leu Asp LysIle Val Ile Gln Asn 180 185 190 ata gga gaa tgt ggt ttt atc gat gga ggggat caa ttg tta att caa 624 Ile Gly Glu Cys Gly Phe Ile Asp Gly Gly AspGln Leu Leu Ile Gln 195 200 205 aag tgc aat agt gta ttt ata gaa aga atggtt tca aag caa agt cag 672 Lys Cys Asn Ser Val Phe Ile Glu Arg Met ValSer Lys Gln Ser Gln 210 215 220 tgg att gat cct gaa aaa act gtg aaa gaattg tat tct gaa cag caa 720 Trp Ile Asp Pro Glu Lys Thr Val Lys Glu LeuTyr Ser Glu Gln Gln 225 230 235 240 att ata ccc aaa cat ata aaa atc ttacaa gca gaa tta ctt caa tgg 768 Ile Ile Pro Lys His Ile Lys Ile Leu GlnAla Glu Leu Leu Gln Trp 245 250 255 cat ttt cct gct tta aaa tat cat agaaat gat tat caa aaa cat ttg 816 His Phe Pro Ala Leu Lys Tyr His Arg AsnAsp Tyr Gln Lys His Leu 260 265 270 gat gct tta act tta gaa gat ttt aaaaaa atc cat tat aga cat aga 864 Asp Ala Leu Thr Leu Glu Asp Phe Lys LysIle His Tyr Arg His Arg 275 280 285 aaa ata aag aaa ata aat tat aca atgctt gat gaa aaa gta att cgt 912 Lys Ile Lys Lys Ile Asn Tyr Thr Met LeuAsp Glu Lys Val Ile Arg 290 295 300 gaa ata tta gat aaa ttt aaa ttg agtggt aaa aaa atg act tta gct 960 Glu Ile Leu Asp Lys Phe Lys Leu Ser GlyLys Lys Met Thr Leu Ala 305 310 315 320 ata ata cct gct cga gct ggt tcaaaa ggt ata aaa aat aaa aat tta 1008 Ile Ile Pro Ala Arg Ala Gly Ser LysGly Ile Lys Asn Lys Asn Leu 325 330 335 gct ctt ttg cat gat agg cct ttgttg tat tat act atc aat gca gca 1056 Ala Leu Leu His Asp Arg Pro Leu LeuTyr Tyr Thr Ile Asn Ala Ala 340 345 350 aaa aat tca aag tat gta gat aaaatt gtt tta agt agt gat ggc gat 1104 Lys Asn Ser Lys Tyr Val Asp Lys IleVal Leu Ser Ser Asp Gly Asp 355 360 365 gat ata tta gaa tat gga caa actcaa ggt gta gat gtg tta aaa aga 1152 Asp Ile Leu Glu Tyr Gly Gln Thr GlnGly Val Asp Val Leu Lys Arg 370 375 380 cct aaa gaa tta gcg cta gat gataca act agt gat aag gtt gta ttg 1200 Pro Lys Glu Leu Ala Leu Asp Asp ThrThr Ser Asp Lys Val Val Leu 385 390 395 400 cat acc ttg agt ttt tat aaagat tat gaa aat att gtt tta tta caa 1248 His Thr Leu Ser Phe Tyr Lys AspTyr Glu Asn Ile Val Leu Leu Gln 405 410 415 ccc act tct cct tta agg acaaat gta cat ata gat gaa gct ttt tta 1296 Pro Thr Ser Pro Leu Arg Thr AsnVal His Ile Asp Glu Ala Phe Leu 420 425 430 aaa ttt aaa aat gaa aac tcaaat gca tta ata agt gtt gta gaa tgt 1344 Lys Phe Lys Asn Glu Asn Ser AsnAla Leu Ile Ser Val Val Glu Cys 435 440 445 gat aat aaa att tta aaa gctttt ata gat gat aat ggt aac tta aaa 1392 Asp Asn Lys Ile Leu Lys Ala PheIle Asp Asp Asn Gly Asn Leu Lys 450 455 460 gga att tgt gat aac aaa tatcca ttt atg cct aga caa aaa tta cca 1440 Gly Ile Cys Asp Asn Lys Tyr ProPhe Met Pro Arg Gln Lys Leu Pro 465 470 475 480 aaa act tat atg agt aatggt gca att tat ata gta aag tca aat tta 1488 Lys Thr Tyr Met Ser Asn GlyAla Ile Tyr Ile Val Lys Ser Asn Leu 485 490 495 ttt tta aat aac cca actttt cta caa gaa aaa aca agt tgc tat ata 1536 Phe Leu Asn Asn Pro Thr PheLeu Gln Glu Lys Thr Ser Cys Tyr Ile 500 505 510 atg gac gaa aaa gct agtttg gat ata gat aca aca gag gat tta aaa 1584 Met Asp Glu Lys Ala Ser LeuAsp Ile Asp Thr Thr Glu Asp Leu Lys 515 520 525 aga gtt aat aat ata agcttc tta 1608 Arg Val Asn Asn Ile Ser Phe Leu 530 535 19 536 PRTCampylobacter jejuni beta-1,4 N-acetylgalactosaminyl (GalNAc) 19 Met ThrLeu Phe Tyr Lys Ile Ile Ala Phe Leu Arg Leu Leu Lys Ile 1 5 10 15 AspLys Lys Leu Lys Phe Asp Asn Glu Tyr Phe Leu Asn Leu Asn Lys 20 25 30 LysIle Tyr Asn Glu Lys His Lys Gly Phe Phe Asp Phe Asp Pro Asn 35 40 45 SerLys Asp Thr Lys Ser Pro Leu Asn Pro Trp Ala Phe Ile Arg Val 50 55 60 LysAsn Glu Ala Thr Thr Leu Arg Val Ser Leu Glu Ser Met Leu Pro 65 70 75 80Ala Ile Gln Arg Gly Val Ile Gly Tyr Asn Asp Cys Thr Asp Gly Ser 85 90 95Glu Glu Ile Ile Leu Glu Phe Cys Lys Gln Tyr Pro Ser Phe Ile Pro 100 105110 Val Lys Tyr Pro His Glu Val Gln Ile Glu Asn Pro Gln Ser Glu Glu 115120 125 Asn Lys Leu His Ser Tyr Tyr Asn Tyr Val Ala Ser Phe Ile Pro Gln130 135 140 Asp Glu Trp Leu Ile Lys Ile Asp Val Asp His Tyr Tyr Asp AlaLys 145 150 155 160 Lys Leu Tyr Lys Ser Phe Tyr Met Ala Ser Lys Asn ThrAla Val Arg 165 170 175 Phe Pro Arg Ile Asn Phe Leu Ile Leu Asp Lys IleVal Ile Gln Asn 180 185 190 Ile Gly Glu Cys Gly Phe Ile Asp Gly Gly AspGln Leu Leu Ile Gln 195 200 205 Lys Cys Asn Ser Val Phe Ile Glu Arg MetVal Ser Lys Gln Ser Gln 210 215 220 Trp Ile Asp Pro Glu Lys Thr Val LysGlu Leu Tyr Ser Glu Gln Gln 225 230 235 240 Ile Ile Pro Lys His Ile LysIle Leu Gln Ala Glu Leu Leu Gln Trp 245 250 255 His Phe Pro Ala Leu LysTyr His Arg Asn Asp Tyr Gln Lys His Leu 260 265 270 Asp Ala Leu Thr LeuGlu Asp Phe Lys Lys Ile His Tyr Arg His Arg 275 280 285 Lys Ile Lys LysIle Asn Tyr Thr Met Leu Asp Glu Lys Val Ile Arg 290 295 300 Glu Ile LeuAsp Lys Phe Lys Leu Ser Gly Lys Lys Met Thr Leu Ala 305 310 315 320 IleIle Pro Ala Arg Ala Gly Ser Lys Gly Ile Lys Asn Lys Asn Leu 325 330 335Ala Leu Leu His Asp Arg Pro Leu Leu Tyr Tyr Thr Ile Asn Ala Ala 340 345350 Lys Asn Ser Lys Tyr Val Asp Lys Ile Val Leu Ser Ser Asp Gly Asp 355360 365 Asp Ile Leu Glu Tyr Gly Gln Thr Gln Gly Val Asp Val Leu Lys Arg370 375 380 Pro Lys Glu Leu Ala Leu Asp Asp Thr Thr Ser Asp Lys Val ValLeu 385 390 395 400 His Thr Leu Ser Phe Tyr Lys Asp Tyr Glu Asn Ile ValLeu Leu Gln 405 410 415 Pro Thr Ser Pro Leu Arg Thr Asn Val His Ile AspGlu Ala Phe Leu 420 425 430 Lys Phe Lys Asn Glu Asn Ser Asn Ala Leu IleSer Val Val Glu Cys 435 440 445 Asp Asn Lys Ile Leu Lys Ala Phe Ile AspAsp Asn Gly Asn Leu Lys 450 455 460 Gly Ile Cys Asp Asn Lys Tyr Pro PheMet Pro Arg Gln Lys Leu Pro 465 470 475 480 Lys Thr Tyr Met Ser Asn GlyAla Ile Tyr Ile Val Lys Ser Asn Leu 485 490 495 Phe Leu Asn Asn Pro ThrPhe Leu Gln Glu Lys Thr Ser Cys Tyr Ile 500 505 510 Met Asp Glu Lys AlaSer Leu Asp Ile Asp Thr Thr Glu Asp Leu Lys 515 520 525 Arg Val Asn AsnIle Ser Phe Leu 530 535 20 1056 DNA Campylobacter jejuni CDS (1)..(1056)beta-1,4 N-acetylgalactosaminyl (GalNAc) transferase from C. jejuni O1020 atg cta ttt caa tca tac ttt gtg aaa ata att tgc tta ttc atc cct 48Met Leu Phe Gln Ser Tyr Phe Val Lys Ile Ile Cys Leu Phe Ile Pro 1 5 1015 ttt aga aaa att aga cat aaa ata aaa aaa aca ttt tta cta aaa aac 96Phe Arg Lys Ile Arg His Lys Ile Lys Lys Thr Phe Leu Leu Lys Asn 20 25 30ata caa cga gat aaa atc gat tct tat cta cca aaa aaa act ctt ata 144 IleGln Arg Asp Lys Ile Asp Ser Tyr Leu Pro Lys Lys Thr Leu Ile 35 40 45 caaatt aat aaa tac aac aat gaa gat tta att aaa ctt aat aaa gct 192 Gln IleAsn Lys Tyr Asn Asn Glu Asp Leu Ile Lys Leu Asn Lys Ala 50 55 60 att ataggg ggg ggg cat aaa gga tat ttt aat tat gat gaa aaa tct 240 Ile Ile GlyGly Gly His Lys Gly Tyr Phe Asn Tyr Asp Glu Lys Ser 65 70 75 80 aaa gatcca aaa tct cct ttg aat cct tgg gct ttt ata cga gta aaa 288 Lys Asp ProLys Ser Pro Leu Asn Pro Trp Ala Phe Ile Arg Val Lys 85 90 95 aat gaa gctatt acc tta aaa gct tct ctt gaa agc ata ttg cct gct 336 Asn Glu Ala IleThr Leu Lys Ala Ser Leu Glu Ser Ile Leu Pro Ala 100 105 110 att caa agaggt gtt ata gga tat aat gat tgc acc gat gga agt gaa 384 Ile Gln Arg GlyVal Ile Gly Tyr Asn Asp Cys Thr Asp Gly Ser Glu 115 120 125 gaa ata attcta gaa ttt tgc aaa caa tat cct tca ttt ata cca ata 432 Glu Ile Ile LeuGlu Phe Cys Lys Gln Tyr Pro Ser Phe Ile Pro Ile 130 135 140 aaa tat ccttat gaa att caa att caa aac cca aaa tca gaa gaa aat 480 Lys Tyr Pro TyrGlu Ile Gln Ile Gln Asn Pro Lys Ser Glu Glu Asn 145 150 155 160 aaa ctctat agc tat tat aat tat gtt gca agt ttt ata cca aaa gat 528 Lys Leu TyrSer Tyr Tyr Asn Tyr Val Ala Ser Phe Ile Pro Lys Asp 165 170 175 gag tggctc ata aaa ata gat gtg gat cat tat tat gat gca aaa aaa 576 Glu Trp LeuIle Lys Ile Asp Val Asp His Tyr Tyr Asp Ala Lys Lys 180 185 190 tta tataag agt ttt tat ata cct aga aaa aat tat cat gta att agt 624 Leu Tyr LysSer Phe Tyr Ile Pro Arg Lys Asn Tyr His Val Ile Ser 195 200 205 tac tctagg ata gat ttt ata ttt aat gaa gaa aaa ttt tat gtt tat 672 Tyr Ser ArgIle Asp Phe Ile Phe Asn Glu Glu Lys Phe Tyr Val Tyr 210 215 220 cgg aataag gag ggg gag att tta aaa gct cct gga gat tgt tta gca 720 Arg Asn LysGlu Gly Glu Ile Leu Lys Ala Pro Gly Asp Cys Leu Ala 225 230 235 240 atacaa aac act aac tta ttt tgg aaa gaa ata ctt att gaa gat gat 768 Ile GlnAsn Thr Asn Leu Phe Trp Lys Glu Ile Leu Ile Glu Asp Asp 245 250 255 acattt aag tgg aat act gca aaa aat aat ata gag aat gca aaa tca 816 Thr PheLys Trp Asn Thr Ala Lys Asn Asn Ile Glu Asn Ala Lys Ser 260 265 270 tatgaa att tta aaa gtt aga aat aga att tat ttt act aca gaa ctt 864 Tyr GluIle Leu Lys Val Arg Asn Arg Ile Tyr Phe Thr Thr Glu Leu 275 280 285 aataat tat cat ttt cca ttt ata aaa aat tat aga aaa aat gat tat 912 Asn AsnTyr His Phe Pro Phe Ile Lys Asn Tyr Arg Lys Asn Asp Tyr 290 295 300 aagcag tta aat tgg gtt agc tta gat gat ttt att aaa aat tat aaa 960 Lys GlnLeu Asn Trp Val Ser Leu Asp Asp Phe Ile Lys Asn Tyr Lys 305 310 315 320gaa aaa tta aaa aat caa ata gat ttt aaa atg cta gaa tac aaa aca 1008 GluLys Leu Lys Asn Gln Ile Asp Phe Lys Met Leu Glu Tyr Lys Thr 325 330 335tta aaa aaa gtg tac aaa aag ctt aca tct tca gca agc gat aaa att 1056 LeuLys Lys Val Tyr Lys Lys Leu Thr Ser Ser Ala Ser Asp Lys Ile 340 345 35021 352 PRT Campylobacter jejuni beta-1,4 N-acetylgalactosaminyl (GalNAc)transferase from C. jejuni O10 21 Met Leu Phe Gln Ser Tyr Phe Val LysIle Ile Cys Leu Phe Ile Pro 1 5 10 15 Phe Arg Lys Ile Arg His Lys IleLys Lys Thr Phe Leu Leu Lys Asn 20 25 30 Ile Gln Arg Asp Lys Ile Asp SerTyr Leu Pro Lys Lys Thr Leu Ile 35 40 45 Gln Ile Asn Lys Tyr Asn Asn GluAsp Leu Ile Lys Leu Asn Lys Ala 50 55 60 Ile Ile Gly Gly Gly His Lys GlyTyr Phe Asn Tyr Asp Glu Lys Ser 65 70 75 80 Lys Asp Pro Lys Ser Pro LeuAsn Pro Trp Ala Phe Ile Arg Val Lys 85 90 95 Asn Glu Ala Ile Thr Leu LysAla Ser Leu Glu Ser Ile Leu Pro Ala 100 105 110 Ile Gln Arg Gly Val IleGly Tyr Asn Asp Cys Thr Asp Gly Ser Glu 115 120 125 Glu Ile Ile Leu GluPhe Cys Lys Gln Tyr Pro Ser Phe Ile Pro Ile 130 135 140 Lys Tyr Pro TyrGlu Ile Gln Ile Gln Asn Pro Lys Ser Glu Glu Asn 145 150 155 160 Lys LeuTyr Ser Tyr Tyr Asn Tyr Val Ala Ser Phe Ile Pro Lys Asp 165 170 175 GluTrp Leu Ile Lys Ile Asp Val Asp His Tyr Tyr Asp Ala Lys Lys 180 185 190Leu Tyr Lys Ser Phe Tyr Ile Pro Arg Lys Asn Tyr His Val Ile Ser 195 200205 Tyr Ser Arg Ile Asp Phe Ile Phe Asn Glu Glu Lys Phe Tyr Val Tyr 210215 220 Arg Asn Lys Glu Gly Glu Ile Leu Lys Ala Pro Gly Asp Cys Leu Ala225 230 235 240 Ile Gln Asn Thr Asn Leu Phe Trp Lys Glu Ile Leu Ile GluAsp Asp 245 250 255 Thr Phe Lys Trp Asn Thr Ala Lys Asn Asn Ile Glu AsnAla Lys Ser 260 265 270 Tyr Glu Ile Leu Lys Val Arg Asn Arg Ile Tyr PheThr Thr Glu Leu 275 280 285 Asn Asn Tyr His Phe Pro Phe Ile Lys Asn TyrArg Lys Asn Asp Tyr 290 295 300 Lys Gln Leu Asn Trp Val Ser Leu Asp AspPhe Ile Lys Asn Tyr Lys 305 310 315 320 Glu Lys Leu Lys Asn Gln Ile AspPhe Lys Met Leu Glu Tyr Lys Thr 325 330 335 Leu Lys Lys Val Tyr Lys LysLeu Thr Ser Ser Ala Ser Asp Lys Ile 340 345 350 22 945 DNA Campylobacterjejuni CDS (1)..(945) beta-1,4 N-acetylgalactosaminyl (GalNAc)transferase from C. jejuni O36 22 atg ctt aaa aaa atc att tct tta tataaa aga tac tcg att tct aaa 48 Met Leu Lys Lys Ile Ile Ser Leu Tyr LysArg Tyr Ser Ile Ser Lys 1 5 10 15 aaa ttg gtt tta gat aat gag cat ttcatt aag gaa aat aaa aac atc 96 Lys Leu Val Leu Asp Asn Glu His Phe IleLys Glu Asn Lys Asn Ile 20 25 30 tat gga aaa aaa cat aag ggc ttt ttt gacttt gat gaa aag gct aag 144 Tyr Gly Lys Lys His Lys Gly Phe Phe Asp PheAsp Glu Lys Ala Lys 35 40 45 gat gtg aaa tca ccc ctt aat cct tgg gga tttatc agg gtt aaa aat 192 Asp Val Lys Ser Pro Leu Asn Pro Trp Gly Phe IleArg Val Lys Asn 50 55 60 gaa gct tta acc cta aga gtt tct tta gaa agt atacta cct gct tta 240 Glu Ala Leu Thr Leu Arg Val Ser Leu Glu Ser Ile LeuPro Ala Leu 65 70 75 80 caa aga gga att ata gct tac aac gac tgt gat gatggg agt gaa gag 288 Gln Arg Gly Ile Ile Ala Tyr Asn Asp Cys Asp Asp GlySer Glu Glu 85 90 95 ctt att tta gaa ttt tgc aag caa tat ccc aac ttc attgct aaa aaa 336 Leu Ile Leu Glu Phe Cys Lys Gln Tyr Pro Asn Phe Ile AlaLys Lys 100 105 110 tat cct tat aaa gta gat cta gaa aat cct aaa aat gaagaa aat aaa 384 Tyr Pro Tyr Lys Val Asp Leu Glu Asn Pro Lys Asn Glu GluAsn Lys 115 120 125 ctt tac tct tat tac aat tgg gca gca tct ttt ata ccctta gat gag 432 Leu Tyr Ser Tyr Tyr Asn Trp Ala Ala Ser Phe Ile Pro LeuAsp Glu 130 135 140 tgg ttt ata aaa atc gat gtg gat cat tac tac gat gccaag aag ctt 480 Trp Phe Ile Lys Ile Asp Val Asp His Tyr Tyr Asp Ala LysLys Leu 145 150 155 160 tat aag agt ttt tat agg att gat caa gaa aat aaagcc tta tgc tac 528 Tyr Lys Ser Phe Tyr Arg Ile Asp Gln Glu Asn Lys AlaLeu Cys Tyr 165 170 175 cca aga att aat ttt ata atc tta aat gga aat atttat gtg caa aat 576 Pro Arg Ile Asn Phe Ile Ile Leu Asn Gly Asn Ile TyrVal Gln Asn 180 185 190 agt gga aat tat gga ttc ata ggg ggg ggg gat caactc ttg att aaa 624 Ser Gly Asn Tyr Gly Phe Ile Gly Gly Gly Asp Gln LeuLeu Ile Lys 195 200 205 aga aga aat agt agc ttt ata gaa aga agg gtt tcaaaa aaa agc caa 672 Arg Arg Asn Ser Ser Phe Ile Glu Arg Arg Val Ser LysLys Ser Gln 210 215 220 tgg ata gat cct aag gga ctt ata gaa gaa ctc tactcc gag caa caa 720 Trp Ile Asp Pro Lys Gly Leu Ile Glu Glu Leu Tyr SerGlu Gln Gln 225 230 235 240 gtc tta tct caa gga gtg aaa ata cta caa gctccc cta ctt cag tgg 768 Val Leu Ser Gln Gly Val Lys Ile Leu Gln Ala ProLeu Leu Gln Trp 245 250 255 cat ttt cct gcc tta aaa tac cgc cga aac gattac caa caa tat tta 816 His Phe Pro Ala Leu Lys Tyr Arg Arg Asn Asp TyrGln Gln Tyr Leu 260 265 270 gat atc ttg agt tta gaa gaa ttt cag gcc tttcat cgt aag agc aaa 864 Asp Ile Leu Ser Leu Glu Glu Phe Gln Ala Phe HisArg Lys Ser Lys 275 280 285 gag gct aaa aaa ata gac ttt gcc atg cta aaacgc cct gta atc gag 912 Glu Ala Lys Lys Ile Asp Phe Ala Met Leu Lys ArgPro Val Ile Glu 290 295 300 caa ata tta aag aaa ttt caa gga gag ata aaa945 Gln Ile Leu Lys Lys Phe Gln Gly Glu Ile Lys 305 310 315 23 315 PRTCampylobacter jejuni beta-1,4 N-acetylgalactosaminyl (GalNAc)transferase from C. jejuni O36 23 Met Leu Lys Lys Ile Ile Ser Leu TyrLys Arg Tyr Ser Ile Ser Lys 1 5 10 15 Lys Leu Val Leu Asp Asn Glu HisPhe Ile Lys Glu Asn Lys Asn Ile 20 25 30 Tyr Gly Lys Lys His Lys Gly PhePhe Asp Phe Asp Glu Lys Ala Lys 35 40 45 Asp Val Lys Ser Pro Leu Asn ProTrp Gly Phe Ile Arg Val Lys Asn 50 55 60 Glu Ala Leu Thr Leu Arg Val SerLeu Glu Ser Ile Leu Pro Ala Leu 65 70 75 80 Gln Arg Gly Ile Ile Ala TyrAsn Asp Cys Asp Asp Gly Ser Glu Glu 85 90 95 Leu Ile Leu Glu Phe Cys LysGln Tyr Pro Asn Phe Ile Ala Lys Lys 100 105 110 Tyr Pro Tyr Lys Val AspLeu Glu Asn Pro Lys Asn Glu Glu Asn Lys 115 120 125 Leu Tyr Ser Tyr TyrAsn Trp Ala Ala Ser Phe Ile Pro Leu Asp Glu 130 135 140 Trp Phe Ile LysIle Asp Val Asp His Tyr Tyr Asp Ala Lys Lys Leu 145 150 155 160 Tyr LysSer Phe Tyr Arg Ile Asp Gln Glu Asn Lys Ala Leu Cys Tyr 165 170 175 ProArg Ile Asn Phe Ile Ile Leu Asn Gly Asn Ile Tyr Val Gln Asn 180 185 190Ser Gly Asn Tyr Gly Phe Ile Gly Gly Gly Asp Gln Leu Leu Ile Lys 195 200205 Arg Arg Asn Ser Ser Phe Ile Glu Arg Arg Val Ser Lys Lys Ser Gln 210215 220 Trp Ile Asp Pro Lys Gly Leu Ile Glu Glu Leu Tyr Ser Glu Gln Gln225 230 235 240 Val Leu Ser Gln Gly Val Lys Ile Leu Gln Ala Pro Leu LeuGln Trp 245 250 255 His Phe Pro Ala Leu Lys Tyr Arg Arg Asn Asp Tyr GlnGln Tyr Leu 260 265 270 Asp Ile Leu Ser Leu Glu Glu Phe Gln Ala Phe HisArg Lys Ser Lys 275 280 285 Glu Ala Lys Lys Ile Asp Phe Ala Met Leu LysArg Pro Val Ile Glu 290 295 300 Gln Ile Leu Lys Lys Phe Gln Gly Glu IleLys 305 310 315 24 1608 DNA Campylobacter jejuni CDS (1)..(1608)beta-1,4 N-acetylgalactosaminyl (GalNAc) transferase from C. jejuni NCTC11168 24 atg act ttg ttt tat aaa att ata gct ttt tta aga ttg ctt aaa att48 Met Thr Leu Phe Tyr Lys Ile Ile Ala Phe Leu Arg Leu Leu Lys Ile 1 510 15 gat aaa aaa tta aaa ttt gat aat gaa tat ttt tta aac tta aat aaa 96Asp Lys Lys Leu Lys Phe Asp Asn Glu Tyr Phe Leu Asn Leu Asn Lys 20 25 30aaa atc tac gat gaa aag cat aaa ggt ttt ttt gat ttt gat cca aac 144 LysIle Tyr Asp Glu Lys His Lys Gly Phe Phe Asp Phe Asp Pro Asn 35 40 45 tcaaaa gat aca aaa tct cct tta aat cca tgg gct ttt ata aga gta 192 Ser LysAsp Thr Lys Ser Pro Leu Asn Pro Trp Ala Phe Ile Arg Val 50 55 60 aaa aatgaa gcc act act tta aga gta tca ctt gaa agt atg tta cct 240 Lys Asn GluAla Thr Thr Leu Arg Val Ser Leu Glu Ser Met Leu Pro 65 70 75 80 gcc atacaa aga ggt gtt ata gga tat aat gat tgt act gat gga agt 288 Ala Ile GlnArg Gly Val Ile Gly Tyr Asn Asp Cys Thr Asp Gly Ser 85 90 95 gaa gaa attatt ttg gaa ttt tgc aaa caa tac cct tcg ttt ata cca 336 Glu Glu Ile IleLeu Glu Phe Cys Lys Gln Tyr Pro Ser Phe Ile Pro 100 105 110 gta aaa tatccc cat gag gtg caa att gaa aat ccg caa agc gaa gaa 384 Val Lys Tyr ProHis Glu Val Gln Ile Glu Asn Pro Gln Ser Glu Glu 115 120 125 aat aaa cttcat agt tat tat aac tat gta gct agt ttt ata ccg caa 432 Asn Lys Leu HisSer Tyr Tyr Asn Tyr Val Ala Ser Phe Ile Pro Gln 130 135 140 gat gag tggctt ata aaa ata gat gtg gat cat tac tat gat gca aaa 480 Asp Glu Trp LeuIle Lys Ile Asp Val Asp His Tyr Tyr Asp Ala Lys 145 150 155 160 aaa ttatat aag agt ttt tat atg gca tca aaa aat act gct gtt aga 528 Lys Leu TyrLys Ser Phe Tyr Met Ala Ser Lys Asn Thr Ala Val Arg 165 170 175 ttt ccaaga att aat ttt tta ata cta gat aaa att gta att caa aat 576 Phe Pro ArgIle Asn Phe Leu Ile Leu Asp Lys Ile Val Ile Gln Asn 180 185 190 ata ggagaa tgt ggt ttt atc gat gga ggg gat caa ttg tta att caa 624 Ile Gly GluCys Gly Phe Ile Asp Gly Gly Asp Gln Leu Leu Ile Gln 195 200 205 aag tgcaat agt gta ttt ata gaa aga atg gtt tca aag caa agt cag 672 Lys Cys AsnSer Val Phe Ile Glu Arg Met Val Ser Lys Gln Ser Gln 210 215 220 tgg attgat cct gaa aaa act gtg aaa gaa ttg tat tct gaa cag caa 720 Trp Ile AspPro Glu Lys Thr Val Lys Glu Leu Tyr Ser Glu Gln Gln 225 230 235 240 attata ccc aaa cat ata aaa atc tta caa gca gaa tta ctt caa tgg 768 Ile IlePro Lys His Ile Lys Ile Leu Gln Ala Glu Leu Leu Gln Trp 245 250 255 catttt cct gct tta aaa tat cat aga aat gat tat caa aaa cat ttg 816 His PhePro Ala Leu Lys Tyr His Arg Asn Asp Tyr Gln Lys His Leu 260 265 270 gatgct tta act tta gaa gat ttt aaa aaa atc cat tat aga cat aga 864 Asp AlaLeu Thr Leu Glu Asp Phe Lys Lys Ile His Tyr Arg His Arg 275 280 285 aaaata aag aaa ata aat tat aca atg ctt gat gaa aaa gta att cgt 912 Lys IleLys Lys Ile Asn Tyr Thr Met Leu Asp Glu Lys Val Ile Arg 290 295 300 gaaata tta gat aaa ttt aaa ttg agt ggt aaa aaa atg act tta gct 960 Glu IleLeu Asp Lys Phe Lys Leu Ser Gly Lys Lys Met Thr Leu Ala 305 310 315 320ata ata cct gct cga gct ggt tca aaa ggt ata aaa aat aaa aat tta 1008 IleIle Pro Ala Arg Ala Gly Ser Lys Gly Ile Lys Asn Lys Asn Leu 325 330 335gct ctt ttg cat gat agg cct ttg ttg tat tat act atc aat gca gca 1056 AlaLeu Leu His Asp Arg Pro Leu Leu Tyr Tyr Thr Ile Asn Ala Ala 340 345 350aaa aat tca aag tat gta gat aaa att gtt tta agt agt gat ggc gat 1104 LysAsn Ser Lys Tyr Val Asp Lys Ile Val Leu Ser Ser Asp Gly Asp 355 360 365gat ata tta gaa tat gga caa act caa ggt gta gat gtg tta aaa aga 1152 AspIle Leu Glu Tyr Gly Gln Thr Gln Gly Val Asp Val Leu Lys Arg 370 375 380cct aaa gaa tta gcg cta gat gat aca act agt gat aag gtt gta ttg 1200 ProLys Glu Leu Ala Leu Asp Asp Thr Thr Ser Asp Lys Val Val Leu 385 390 395400 cat acc ttg agt ttt tat aaa gat tat gaa aat att gtt tta tta caa 1248His Thr Leu Ser Phe Tyr Lys Asp Tyr Glu Asn Ile Val Leu Leu Gln 405 410415 ccc act tct cct tta agg aca aat gta cat ata gat gaa gct ttt tta 1296Pro Thr Ser Pro Leu Arg Thr Asn Val His Ile Asp Glu Ala Phe Leu 420 425430 aaa ttt aaa aat gaa aac tca aat gca tta ata agt gtt gta gaa tgt 1344Lys Phe Lys Asn Glu Asn Ser Asn Ala Leu Ile Ser Val Val Glu Cys 435 440445 gat aat aaa att tta aaa gct ttt ata gat gat aat ggt aac tta aaa 1392Asp Asn Lys Ile Leu Lys Ala Phe Ile Asp Asp Asn Gly Asn Leu Lys 450 455460 gga att tgt gat aac aaa tat cca ttt atg cct aga caa aaa tta cca 1440Gly Ile Cys Asp Asn Lys Tyr Pro Phe Met Pro Arg Gln Lys Leu Pro 465 470475 480 aaa act tat atg agt aat ggt gca att tat ata gta aag tca aat tta1488 Lys Thr Tyr Met Ser Asn Gly Ala Ile Tyr Ile Val Lys Ser Asn Leu 485490 495 ttt tta aat aac cca act ttt cta caa gaa aaa aca agt tgc tat ata1536 Phe Leu Asn Asn Pro Thr Phe Leu Gln Glu Lys Thr Ser Cys Tyr Ile 500505 510 atg gac gaa aaa gct agt ttg gat ata gat aca aca gag gat tta aaa1584 Met Asp Glu Lys Ala Ser Leu Asp Ile Asp Thr Thr Glu Asp Leu Lys 515520 525 aga gtt aat aat ata agc ttc tta 1608 Arg Val Asn Asn Ile Ser PheLeu 530 535 25 536 PRT Campylobacter jejuni beta-1,4N-acetylgalactosaminyl (GalNAc) transferase from C. jejuni NCTC 11168 25Met Thr Leu Phe Tyr Lys Ile Ile Ala Phe Leu Arg Leu Leu Lys Ile 1 5 1015 Asp Lys Lys Leu Lys Phe Asp Asn Glu Tyr Phe Leu Asn Leu Asn Lys 20 2530 Lys Ile Tyr Asp Glu Lys His Lys Gly Phe Phe Asp Phe Asp Pro Asn 35 4045 Ser Lys Asp Thr Lys Ser Pro Leu Asn Pro Trp Ala Phe Ile Arg Val 50 5560 Lys Asn Glu Ala Thr Thr Leu Arg Val Ser Leu Glu Ser Met Leu Pro 65 7075 80 Ala Ile Gln Arg Gly Val Ile Gly Tyr Asn Asp Cys Thr Asp Gly Ser 8590 95 Glu Glu Ile Ile Leu Glu Phe Cys Lys Gln Tyr Pro Ser Phe Ile Pro100 105 110 Val Lys Tyr Pro His Glu Val Gln Ile Glu Asn Pro Gln Ser GluGlu 115 120 125 Asn Lys Leu His Ser Tyr Tyr Asn Tyr Val Ala Ser Phe IlePro Gln 130 135 140 Asp Glu Trp Leu Ile Lys Ile Asp Val Asp His Tyr TyrAsp Ala Lys 145 150 155 160 Lys Leu Tyr Lys Ser Phe Tyr Met Ala Ser LysAsn Thr Ala Val Arg 165 170 175 Phe Pro Arg Ile Asn Phe Leu Ile Leu AspLys Ile Val Ile Gln Asn 180 185 190 Ile Gly Glu Cys Gly Phe Ile Asp GlyGly Asp Gln Leu Leu Ile Gln 195 200 205 Lys Cys Asn Ser Val Phe Ile GluArg Met Val Ser Lys Gln Ser Gln 210 215 220 Trp Ile Asp Pro Glu Lys ThrVal Lys Glu Leu Tyr Ser Glu Gln Gln 225 230 235 240 Ile Ile Pro Lys HisIle Lys Ile Leu Gln Ala Glu Leu Leu Gln Trp 245 250 255 His Phe Pro AlaLeu Lys Tyr His Arg Asn Asp Tyr Gln Lys His Leu 260 265 270 Asp Ala LeuThr Leu Glu Asp Phe Lys Lys Ile His Tyr Arg His Arg 275 280 285 Lys IleLys Lys Ile Asn Tyr Thr Met Leu Asp Glu Lys Val Ile Arg 290 295 300 GluIle Leu Asp Lys Phe Lys Leu Ser Gly Lys Lys Met Thr Leu Ala 305 310 315320 Ile Ile Pro Ala Arg Ala Gly Ser Lys Gly Ile Lys Asn Lys Asn Leu 325330 335 Ala Leu Leu His Asp Arg Pro Leu Leu Tyr Tyr Thr Ile Asn Ala Ala340 345 350 Lys Asn Ser Lys Tyr Val Asp Lys Ile Val Leu Ser Ser Asp GlyAsp 355 360 365 Asp Ile Leu Glu Tyr Gly Gln Thr Gln Gly Val Asp Val LeuLys Arg 370 375 380 Pro Lys Glu Leu Ala Leu Asp Asp Thr Thr Ser Asp LysVal Val Leu 385 390 395 400 His Thr Leu Ser Phe Tyr Lys Asp Tyr Glu AsnIle Val Leu Leu Gln 405 410 415 Pro Thr Ser Pro Leu Arg Thr Asn Val HisIle Asp Glu Ala Phe Leu 420 425 430 Lys Phe Lys Asn Glu Asn Ser Asn AlaLeu Ile Ser Val Val Glu Cys 435 440 445 Asp Asn Lys Ile Leu Lys Ala PheIle Asp Asp Asn Gly Asn Leu Lys 450 455 460 Gly Ile Cys Asp Asn Lys TyrPro Phe Met Pro Arg Gln Lys Leu Pro 465 470 475 480 Lys Thr Tyr Met SerAsn Gly Ala Ile Tyr Ile Val Lys Ser Asn Leu 485 490 495 Phe Leu Asn AsnPro Thr Phe Leu Gln Glu Lys Thr Ser Cys Tyr Ile 500 505 510 Met Asp GluLys Ala Ser Leu Asp Ile Asp Thr Thr Glu Asp Leu Lys 515 520 525 Arg ValAsn Asn Ile Ser Phe Leu 530 535 26 906 DNA Campylobacter jejuni CDS(1)..(906) beta-1,3-galactosyltransferase from C. jejuni strain OH4384(ORF 6a of lipooligosaccharide (LOS) biosynthesis locus) 26 atg ttt aaaatt tca atc atc tta cca act tat aat gtg gaa caa tat 48 Met Phe Lys IleSer Ile Ile Leu Pro Thr Tyr Asn Val Glu Gln Tyr 1 5 10 15 ata gca agggca ata gaa agc tgt atc aat cag act ttt aaa gat ata 96 Ile Ala Arg AlaIle Glu Ser Cys Ile Asn Gln Thr Phe Lys Asp Ile 20 25 30 gaa ata att gtagtt gat gat tgt gga aat gat aat agt ata aat ata 144 Glu Ile Ile Val ValAsp Asp Cys Gly Asn Asp Asn Ser Ile Asn Ile 35 40 45 gcc aaa gaa tac tctaaa aaa gac aaa aga ata aaa ata atc cac aat 192 Ala Lys Glu Tyr Ser LysLys Asp Lys Arg Ile Lys Ile Ile His Asn 50 55 60 gaa aaa aac tta ggt ctttta aga gca aga tat gaa ggt gtg aaa gta 240 Glu Lys Asn Leu Gly Leu LeuArg Ala Arg Tyr Glu Gly Val Lys Val 65 70 75 80 gca aac tct cct tat ataatg ttt tta gat cct gat gat tat ttg gaa 288 Ala Asn Ser Pro Tyr Ile MetPhe Leu Asp Pro Asp Asp Tyr Leu Glu 85 90 95 cta aat gct tgt gaa gag tgtata aaa att tta gat gaa cag gat gaa 336 Leu Asn Ala Cys Glu Glu Cys IleLys Ile Leu Asp Glu Gln Asp Glu 100 105 110 gtt gat tta gtg ttt ttc aatgct att gtt gaa agt aat gtt att tca 384 Val Asp Leu Val Phe Phe Asn AlaIle Val Glu Ser Asn Val Ile Ser 115 120 125 tat aaa aag ttt gac ttt aattct ggt ttt tat agc aaa aaa gag ttt 432 Tyr Lys Lys Phe Asp Phe Asn SerGly Phe Tyr Ser Lys Lys Glu Phe 130 135 140 gta aaa aaa att att gca aagaaa aat tta tat tgg act atg tgg ggg 480 Val Lys Lys Ile Ile Ala Lys LysAsn Leu Tyr Trp Thr Met Trp Gly 145 150 155 160 aaa ctt ata aga aag aaattg tat tta gaa gct ttt gcg agt tta aga 528 Lys Leu Ile Arg Lys Lys LeuTyr Leu Glu Ala Phe Ala Ser Leu Arg 165 170 175 ctc gag aaa gat gtt aaaatc aat atg gct gaa gat gta ttg tta tat 576 Leu Glu Lys Asp Val Lys IleAsn Met Ala Glu Asp Val Leu Leu Tyr 180 185 190 tat cca atg tta agt caagct caa aaa ata gca tat atg aac tgt aat 624 Tyr Pro Met Leu Ser Gln AlaGln Lys Ile Ala Tyr Met Asn Cys Asn 195 200 205 tta tat cat tac gtg cctaat aat aat tca att tgt aat act aag aat 672 Leu Tyr His Tyr Val Pro AsnAsn Asn Ser Ile Cys Asn Thr Lys Asn 210 215 220 gaa gtg ctt gtt aaa aataat att caa gag ttg cag ttg gtt tta aac 720 Glu Val Leu Val Lys Asn AsnIle Gln Glu Leu Gln Leu Val Leu Asn 225 230 235 240 tat tta agg caa aattat att tta aac aag tat tgt agc gtt ctc tat 768 Tyr Leu Arg Gln Asn TyrIle Leu Asn Lys Tyr Cys Ser Val Leu Tyr 245 250 255 gtg cta att aaa tatttg cta tat att caa ata tat aaa ata aaa aga 816 Val Leu Ile Lys Tyr LeuLeu Tyr Ile Gln Ile Tyr Lys Ile Lys Arg 260 265 270 aca aaa tta atg gttaca tta tta gct aaa ata aat att tta act tta 864 Thr Lys Leu Met Val ThrLeu Leu Ala Lys Ile Asn Ile Leu Thr Leu 275 280 285 aaa att tta ttt aaatat aaa aaa ttt tta aaa caa tgt taa 906 Lys Ile Leu Phe Lys Tyr Lys LysPhe Leu Lys Gln Cys 290 295 300 27 301 PRT Campylobacter jejunibeta-1,3-galactosyltransferase from C. jejuni strain OH4384 (ORF 6a oflipooligosaccharide (LOS) biosynthesis locus) 27 Met Phe Lys Ile Ser IleIle Leu Pro Thr Tyr Asn Val Glu Gln Tyr 1 5 10 15 Ile Ala Arg Ala IleGlu Ser Cys Ile Asn Gln Thr Phe Lys Asp Ile 20 25 30 Glu Ile Ile Val ValAsp Asp Cys Gly Asn Asp Asn Ser Ile Asn Ile 35 40 45 Ala Lys Glu Tyr SerLys Lys Asp Lys Arg Ile Lys Ile Ile His Asn 50 55 60 Glu Lys Asn Leu GlyLeu Leu Arg Ala Arg Tyr Glu Gly Val Lys Val 65 70 75 80 Ala Asn Ser ProTyr Ile Met Phe Leu Asp Pro Asp Asp Tyr Leu Glu 85 90 95 Leu Asn Ala CysGlu Glu Cys Ile Lys Ile Leu Asp Glu Gln Asp Glu 100 105 110 Val Asp LeuVal Phe Phe Asn Ala Ile Val Glu Ser Asn Val Ile Ser 115 120 125 Tyr LysLys Phe Asp Phe Asn Ser Gly Phe Tyr Ser Lys Lys Glu Phe 130 135 140 ValLys Lys Ile Ile Ala Lys Lys Asn Leu Tyr Trp Thr Met Trp Gly 145 150 155160 Lys Leu Ile Arg Lys Lys Leu Tyr Leu Glu Ala Phe Ala Ser Leu Arg 165170 175 Leu Glu Lys Asp Val Lys Ile Asn Met Ala Glu Asp Val Leu Leu Tyr180 185 190 Tyr Pro Met Leu Ser Gln Ala Gln Lys Ile Ala Tyr Met Asn CysAsn 195 200 205 Leu Tyr His Tyr Val Pro Asn Asn Asn Ser Ile Cys Asn ThrLys Asn 210 215 220 Glu Val Leu Val Lys Asn Asn Ile Gln Glu Leu Gln LeuVal Leu Asn 225 230 235 240 Tyr Leu Arg Gln Asn Tyr Ile Leu Asn Lys TyrCys Ser Val Leu Tyr 245 250 255 Val Leu Ile Lys Tyr Leu Leu Tyr Ile GlnIle Tyr Lys Ile Lys Arg 260 265 270 Thr Lys Leu Met Val Thr Leu Leu AlaLys Ile Asn Ile Leu Thr Leu 275 280 285 Lys Ile Leu Phe Lys Tyr Lys LysPhe Leu Lys Gln Cys 290 295 300 28 912 DNA Campylobacter jejuni CDS(1)..(912) Campylobacter glycosyltransferase B (CgtB) beta-1,3galactosyltransferase from C. jejuni serotype O2 (strain NCTC 11168) 28atg agt caa att tcc atc ata cta cca act tat aat gtg gaa aaa tat 48 MetSer Gln Ile Ser Ile Ile Leu Pro Thr Tyr Asn Val Glu Lys Tyr 1 5 10 15att gct aga gca tta gaa agt tgc att aac caa act ttt aaa gat ata 96 IleAla Arg Ala Leu Glu Ser Cys Ile Asn Gln Thr Phe Lys Asp Ile 20 25 30 gaaatc att gta gta gat gat tgt ggt aat gat aaa agt ata gat ata 144 Glu IleIle Val Val Asp Asp Cys Gly Asn Asp Lys Ser Ile Asp Ile 35 40 45 gct aaagag tat gct agt aaa gat gat aga ata aaa atc ata cat aat 192 Ala Lys GluTyr Ala Ser Lys Asp Asp Arg Ile Lys Ile Ile His Asn 50 55 60 gaa gag aattta aag ctt tta aga gca aga tat gaa ggt gct aaa gta 240 Glu Glu Asn LeuLys Leu Leu Arg Ala Arg Tyr Glu Gly Ala Lys Val 65 70 75 80 gca act tcacct tat atc atg ttt tta gat tct gat gat tat tta gaa 288 Ala Thr Ser ProTyr Ile Met Phe Leu Asp Ser Asp Asp Tyr Leu Glu 85 90 95 ctt aat gct tgcgaa gaa tgt att aaa att ttg gat atg ggt ggg ggg 336 Leu Asn Ala Cys GluGlu Cys Ile Lys Ile Leu Asp Met Gly Gly Gly 100 105 110 ggt aaa att gatttg ttg tgt ttt gaa gct ttt att acc aat gca aaa 384 Gly Lys Ile Asp LeuLeu Cys Phe Glu Ala Phe Ile Thr Asn Ala Lys 115 120 125 aaa tca ata aaaaaa tta aat ata aaa caa gga aaa tac aac aac aaa 432 Lys Ser Ile Lys LysLeu Asn Ile Lys Gln Gly Lys Tyr Asn Asn Lys 130 135 140 gaa ttt aca atgcaa ata ctt aaa act aaa aat cca ttt tgg aca atg 480 Glu Phe Thr Met GlnIle Leu Lys Thr Lys Asn Pro Phe Trp Thr Met 145 150 155 160 tgg gct aaaata atc aaa aaa gat att tat tta aaa gcc ttc aac atg 528 Trp Ala Lys IleIle Lys Lys Asp Ile Tyr Leu Lys Ala Phe Asn Met 165 170 175 tta aat ctcaaa aaa gaa atc aaa ata aat atg gca gaa gat gcc tta 576 Leu Asn Leu LysLys Glu Ile Lys Ile Asn Met Ala Glu Asp Ala Leu 180 185 190 tta tat tatcct ttg aca ata tta tct aat gaa ata ttt tac tta aca 624 Leu Tyr Tyr ProLeu Thr Ile Leu Ser Asn Glu Ile Phe Tyr Leu Thr 195 200 205 caa cct ttgtat acc cag cat gta aat agc aat tct ata aca aat aat 672 Gln Pro Leu TyrThr Gln His Val Asn Ser Asn Ser Ile Thr Asn Asn 210 215 220 att aat tcttta gaa gct aat att caa gaa cat aaa att gtt tta aat 720 Ile Asn Ser LeuGlu Ala Asn Ile Gln Glu His Lys Ile Val Leu Asn 225 230 235 240 gtt ttaaaa tca att aaa aat aaa aaa aca cct cta tat ttt cta att 768 Val Leu LysSer Ile Lys Asn Lys Lys Thr Pro Leu Tyr Phe Leu Ile 245 250 255 ata tattta tta aaa att caa tta ttg aaa tat gaa caa aat ttt aat 816 Ile Tyr LeuLeu Lys Ile Gln Leu Leu Lys Tyr Glu Gln Asn Phe Asn 260 265 270 aaa agaaat ata aat ctt att tat tat aaa ata aat att tta tat caa 864 Lys Arg AsnIle Asn Leu Ile Tyr Tyr Lys Ile Asn Ile Leu Tyr Gln 275 280 285 aaa tatcaa ttc aaa tgg aaa aaa ttt tta tat aat tta att ccg taa 912 Lys Tyr GlnPhe Lys Trp Lys Lys Phe Leu Tyr Asn Leu Ile Pro 290 295 300 29 303 PRTCampylobacter jejuni Campylobacter glycosyltransferase B (CgtB) beta-1,3galactosyltransferase from C. jejuni serotype O2 (strain NCTC 11168) 29Met Ser Gln Ile Ser Ile Ile Leu Pro Thr Tyr Asn Val Glu Lys Tyr 1 5 1015 Ile Ala Arg Ala Leu Glu Ser Cys Ile Asn Gln Thr Phe Lys Asp Ile 20 2530 Glu Ile Ile Val Val Asp Asp Cys Gly Asn Asp Lys Ser Ile Asp Ile 35 4045 Ala Lys Glu Tyr Ala Ser Lys Asp Asp Arg Ile Lys Ile Ile His Asn 50 5560 Glu Glu Asn Leu Lys Leu Leu Arg Ala Arg Tyr Glu Gly Ala Lys Val 65 7075 80 Ala Thr Ser Pro Tyr Ile Met Phe Leu Asp Ser Asp Asp Tyr Leu Glu 8590 95 Leu Asn Ala Cys Glu Glu Cys Ile Lys Ile Leu Asp Met Gly Gly Gly100 105 110 Gly Lys Ile Asp Leu Leu Cys Phe Glu Ala Phe Ile Thr Asn AlaLys 115 120 125 Lys Ser Ile Lys Lys Leu Asn Ile Lys Gln Gly Lys Tyr AsnAsn Lys 130 135 140 Glu Phe Thr Met Gln Ile Leu Lys Thr Lys Asn Pro PheTrp Thr Met 145 150 155 160 Trp Ala Lys Ile Ile Lys Lys Asp Ile Tyr LeuLys Ala Phe Asn Met 165 170 175 Leu Asn Leu Lys Lys Glu Ile Lys Ile AsnMet Ala Glu Asp Ala Leu 180 185 190 Leu Tyr Tyr Pro Leu Thr Ile Leu SerAsn Glu Ile Phe Tyr Leu Thr 195 200 205 Gln Pro Leu Tyr Thr Gln His ValAsn Ser Asn Ser Ile Thr Asn Asn 210 215 220 Ile Asn Ser Leu Glu Ala AsnIle Gln Glu His Lys Ile Val Leu Asn 225 230 235 240 Val Leu Lys Ser IleLys Asn Lys Lys Thr Pro Leu Tyr Phe Leu Ile 245 250 255 Ile Tyr Leu LeuLys Ile Gln Leu Leu Lys Tyr Glu Gln Asn Phe Asn 260 265 270 Lys Arg AsnIle Asn Leu Ile Tyr Tyr Lys Ile Asn Ile Leu Tyr Gln 275 280 285 Lys TyrGln Phe Lys Trp Lys Lys Phe Leu Tyr Asn Leu Ile Pro 290 295 300 30 891DNA Campylobacter jejuni CDS (1)..(891) beta-1,3 galactosyl transferasefrom C. jejuni O10 30 atg ttt aaa att tca atc atc ttg cca act tat aatgtg gaa caa tat 48 Met Phe Lys Ile Ser Ile Ile Leu Pro Thr Tyr Asn ValGlu Gln Tyr 1 5 10 15 ata gca agg gca ata gaa agt tgt atc aat cag actttt aaa aat ata 96 Ile Ala Arg Ala Ile Glu Ser Cys Ile Asn Gln Thr PheLys Asn Ile 20 25 30 gaa ata att gta gtt gat gat tgt gga agt gac aaa agtata gat ata 144 Glu Ile Ile Val Val Asp Asp Cys Gly Ser Asp Lys Ser IleAsp Ile 35 40 45 gtt aaa gaa tat gcc aaa aaa gat gat aga ata aaa atc atacat aat 192 Val Lys Glu Tyr Ala Lys Lys Asp Asp Arg Ile Lys Ile Ile HisAsn 50 55 60 gaa gaa aat tta aaa ctt tta aga gct aga tat gaa ggt gta aaagta 240 Glu Glu Asn Leu Lys Leu Leu Arg Ala Arg Tyr Glu Gly Val Lys Val65 70 75 80 gca aac tct cct tat ata atg ttt tta gat cct gat gat tat ttagaa 288 Ala Asn Ser Pro Tyr Ile Met Phe Leu Asp Pro Asp Asp Tyr Leu Glu85 90 95 ctt aat gct tgt gaa gaa tgt atg aaa att tta aaa aac aat gaa ata336 Leu Asn Ala Cys Glu Glu Cys Met Lys Ile Leu Lys Asn Asn Glu Ile 100105 110 gat tta tta ttt ttt aat gca ttt gta ttg gaa aat aac aat aaa ata384 Asp Leu Leu Phe Phe Asn Ala Phe Val Leu Glu Asn Asn Asn Lys Ile 115120 125 gaa aga aag ttg aat ttt caa gaa aaa tgt tat gta aaa aaa gat ttt432 Glu Arg Lys Leu Asn Phe Gln Glu Lys Cys Tyr Val Lys Lys Asp Phe 130135 140 tta aaa gaa cta tta aaa act aaa aat tta ttt tgg aca gtg tgg gca480 Leu Lys Glu Leu Leu Lys Thr Lys Asn Leu Phe Trp Thr Val Trp Ala 145150 155 160 aaa gtc ata aaa aaa gaa tta tat ctc aag gct gtt ggt tta atatcg 528 Lys Val Ile Lys Lys Glu Leu Tyr Leu Lys Ala Val Gly Leu Ile Ser165 170 175 cta gaa aat gct aaa ata aat atg gct gaa gat gtt tta tta tattac 576 Leu Glu Asn Ala Lys Ile Asn Met Ala Glu Asp Val Leu Leu Tyr Tyr180 185 190 cct ttg ata aat att tca aat act ata ttt cac ttg agt aaa aattta 624 Pro Leu Ile Asn Ile Ser Asn Thr Ile Phe His Leu Ser Lys Asn Leu195 200 205 tac aat tat caa ata aat aat ttc tct ata acc aaa aca tta acattg 672 Tyr Asn Tyr Gln Ile Asn Asn Phe Ser Ile Thr Lys Thr Leu Thr Leu210 215 220 caa aat ata aaa aca aat ata caa gaa caa gat aat gtt cta tatctt 720 Gln Asn Ile Lys Thr Asn Ile Gln Glu Gln Asp Asn Val Leu Tyr Leu225 230 235 240 cta aag aag atg caa tat aat tac aat ttt aac tta act ttgctt aaa 768 Leu Lys Lys Met Gln Tyr Asn Tyr Asn Phe Asn Leu Thr Leu LeuLys 245 250 255 tta att gag tat ttt tta tta att gaa aaa tac tca tta tcaagc aag 816 Leu Ile Glu Tyr Phe Leu Leu Ile Glu Lys Tyr Ser Leu Ser SerLys 260 265 270 cga aat gtt ctt tgt ttt aaa atc aat att ttt ttt aaa aaaatc caa 864 Arg Asn Val Leu Cys Phe Lys Ile Asn Ile Phe Phe Lys Lys IleGln 275 280 285 ttt aaa ttt tat cgc ttg ctg aag atg 891 Phe Lys Phe TyrArg Leu Leu Lys Met 290 295 31 297 PRT Campylobacter jejuni beta-1,3galactosyl transferase from C. jejuni O10 31 Met Phe Lys Ile Ser Ile IleLeu Pro Thr Tyr Asn Val Glu Gln Tyr 1 5 10 15 Ile Ala Arg Ala Ile GluSer Cys Ile Asn Gln Thr Phe Lys Asn Ile 20 25 30 Glu Ile Ile Val Val AspAsp Cys Gly Ser Asp Lys Ser Ile Asp Ile 35 40 45 Val Lys Glu Tyr Ala LysLys Asp Asp Arg Ile Lys Ile Ile His Asn 50 55 60 Glu Glu Asn Leu Lys LeuLeu Arg Ala Arg Tyr Glu Gly Val Lys Val 65 70 75 80 Ala Asn Ser Pro TyrIle Met Phe Leu Asp Pro Asp Asp Tyr Leu Glu 85 90 95 Leu Asn Ala Cys GluGlu Cys Met Lys Ile Leu Lys Asn Asn Glu Ile 100 105 110 Asp Leu Leu PhePhe Asn Ala Phe Val Leu Glu Asn Asn Asn Lys Ile 115 120 125 Glu Arg LysLeu Asn Phe Gln Glu Lys Cys Tyr Val Lys Lys Asp Phe 130 135 140 Leu LysGlu Leu Leu Lys Thr Lys Asn Leu Phe Trp Thr Val Trp Ala 145 150 155 160Lys Val Ile Lys Lys Glu Leu Tyr Leu Lys Ala Val Gly Leu Ile Ser 165 170175 Leu Glu Asn Ala Lys Ile Asn Met Ala Glu Asp Val Leu Leu Tyr Tyr 180185 190 Pro Leu Ile Asn Ile Ser Asn Thr Ile Phe His Leu Ser Lys Asn Leu195 200 205 Tyr Asn Tyr Gln Ile Asn Asn Phe Ser Ile Thr Lys Thr Leu ThrLeu 210 215 220 Gln Asn Ile Lys Thr Asn Ile Gln Glu Gln Asp Asn Val LeuTyr Leu 225 230 235 240 Leu Lys Lys Met Gln Tyr Asn Tyr Asn Phe Asn LeuThr Leu Leu Lys 245 250 255 Leu Ile Glu Tyr Phe Leu Leu Ile Glu Lys TyrSer Leu Ser Ser Lys 260 265 270 Arg Asn Val Leu Cys Phe Lys Ile Asn IlePhe Phe Lys Lys Ile Gln 275 280 285 Phe Lys Phe Tyr Arg Leu Leu Lys Met290 295 32 295 PRT Campylobacter jejuni lipid A biosynthesisacyltransferase from C. jejuni OH4384 32 Met Lys Asn Ser Asp Arg Ile TyrLeu Ser Leu Tyr Tyr Ile Leu Lys 1 5 10 15 Phe Phe Val Thr Phe Met ProAsp Cys Ile Leu His Phe Leu Ala Leu 20 25 30 Ile Val Ala Arg Ile Ala PheHis Leu Asn Lys Lys His Arg Lys Ile 35 40 45 Ile Asn Thr Asn Leu Gln IleCys Phe Pro Gln Tyr Thr Gln Lys Glu 50 55 60 Arg Asp Lys Leu Ser Leu LysIle Tyr Glu Asn Phe Ala Gln Phe Gly 65 70 75 80 Ile Asp Cys Leu Gln AsnGln Asn Thr Thr Lys Glu Lys Ile Leu Asn 85 90 95 Lys Val Asn Phe Ile AsnGlu Asn Phe Leu Ile Asp Ala Leu Ala Leu 100 105 110 Lys Arg Pro Ile IlePhe Thr Thr Ala His Tyr Gly Asn Trp Glu Ile 115 120 125 Leu Ser Leu AlaTyr Ala Ala Lys Tyr Gly Ala Ile Ser Ile Val Gly 130 135 140 Lys Lys LeuLys Ser Glu Val Met Tyr Glu Ile Leu Ser Gln Ser Arg 145 150 155 160 ThrGln Phe Asp Ile Glu Leu Ile Asp Lys Lys Gly Gly Ile Arg Gln 165 170 175Met Leu Ser Ala Leu Lys Lys Glu Arg Ala Leu Gly Ile Leu Thr Asp 180 185190 Gln Asp Cys Val Glu Asn Glu Ser Val Arg Leu Lys Phe Phe Asn Lys 195200 205 Glu Val Asn Tyr Gln Met Gly Ala Ser Leu Ile Ala Gln Arg Ser Asn210 215 220 Ala Leu Ile Ile Pro Val Tyr Ala Tyr Lys Glu Gly Gly Lys PheCys 225 230 235 240 Ile Glu Phe Phe Lys Ala Lys Asp Ser Gln Asn Ala SerLeu Glu Glu 245 250 255 Leu Thr Leu Tyr Gln Ala Gln Ser Cys Glu Glu MetIle Lys Lys Arg 260 265 270 Pro Trp Glu Tyr Phe Phe Phe His Arg Arg PheAla Ser Tyr Asn Glu 275 280 285 Glu Ile Tyr Lys Gly Ala Lys 290 295 33418 PRT Campylobacter jejuni glycosyltransferase from C. jejuni OH4384(ORF 3a of lipooligosaccharide (LOS) biosynthesis locus) 33 Met Asn LeuLys Gln Ile Ser Val Ile Ile Ile Val Lys Asn Ala Glu 1 5 10 15 Gln ThrLeu Leu Glu Cys Leu Asn Ser Leu Lys Asp Phe Asp Glu Ile 20 25 30 Ile LeuLeu Asn Asn Glu Ser Ser Asp Asn Thr Leu Lys Ile Ala Asn 35 40 45 Glu PheLys Lys Asp Phe Ala Asn Leu Tyr Ile Tyr His Asn Ala Phe 50 55 60 Ile GlyPhe Gly Ala Leu Lys Asn Leu Ala Leu Ser Tyr Ala Lys Asn 65 70 75 80 AspTrp Ile Leu Ser Ile Asp Ala Asp Glu Val Leu Glu Asn Glu Cys 85 90 95 IleLys Glu Leu Lys Asn Leu Lys Leu Gln Glu Asp Asn Ile Ile Ala 100 105 110Leu Ser Arg Lys Asn Leu Tyr Lys Gly Glu Trp Ile Lys Ala Cys Gly 115 120125 Trp Trp Pro Asp Tyr Val Leu Arg Ile Phe Asn Lys Asn Phe Thr Arg 130135 140 Phe Asn Asp Asn Leu Val His Glu Ser Leu Val Leu Pro Ser Asn Ala145 150 155 160 Lys Lys Ile Tyr Leu Lys Asn Gly Leu Lys His Tyr Ser TyrLys Asp 165 170 175 Ile Ser His Leu Ile Asp Lys Met Gln Tyr Tyr Ser SerLeu Trp Ala 180 185 190 Lys Gln Asn Ile His Lys Lys Ser Gly Val Leu LysAla Asn Leu Arg 195 200 205 Ala Phe Trp Thr Phe Phe Arg Asn Tyr Phe LeuLys Asn Gly Phe Leu 210 215 220 Tyr Gly Tyr Lys Gly Phe Ile Ile Ser ValCys Ser Ala Leu Gly Thr 225 230 235 240 Phe Phe Lys Tyr Met Lys Leu TyrGlu Leu Gln Arg Gln Lys Pro Lys 245 250 255 Thr Cys Ala Leu Ile Ile IleThr Tyr Asn Gln Lys Glu Arg Leu Lys 260 265 270 Leu Val Leu Asp Ser ValLys Asn Leu Ala Phe Leu Pro Asn Glu Val 275 280 285 Leu Ile Ala Asp AspGly Ser Lys Glu Asp Thr Ala Arg Leu Ile Glu 290 295 300 Glu Tyr Gln LysAsp Phe Pro Cys Pro Leu Lys His Ile Trp Gln Glu 305 310 315 320 Asp GluGly Phe Lys Leu Ser Lys Ser Arg Asn Lys Thr Ile Lys Asn 325 330 335 AlaAsp Ser Glu Tyr Ile Ile Val Ile Asp Gly Asp Met Ile Leu Glu 340 345 350Lys Asp Phe Ile Lys Glu His Leu Glu Phe Ala Gln Arg Lys Leu Phe 355 360365 Leu Gln Gly Ser Arg Val Ile Leu Asn Lys Lys Glu Ser Glu Glu Ile 370375 380 Leu Asn Lys Asp Asp Tyr Arg Ile Ile Phe Asn Lys Lys Asp Phe Lys385 390 395 400 Ser Ser Lys Asn Ser Phe Leu Ala Lys Ile Phe Tyr Ser LeuSer Lys 405 410 415 Lys Arg 34 389 PRT Campylobacter jejuniglycosyltransferase of C. jejuni OH4384 (ORF 4a of lipooligosaccharide(LOS) biosynthesis locus) 34 Met Lys Lys Ile Gly Val Val Ile Pro Ile TyrAsn Val Glu Lys Tyr 1 5 10 15 Leu Arg Glu Cys Leu Asp Ser Val Ile AsnGln Thr Tyr Thr Asn Leu 20 25 30 Glu Ile Ile Leu Val Asn Asp Gly Ser ThrAsp Glu His Ser Leu Asn 35 40 45 Ile Ala Lys Glu Tyr Thr Leu Lys Asp LysArg Ile Thr Leu Phe Asp 50 55 60 Lys Lys Asn Gly Gly Leu Ser Ser Ala ArgAsn Ile Gly Ile Glu Tyr 65 70 75 80 Phe Ser Gly Glu Tyr Lys Leu Lys AsnLys Thr Gln His Ile Lys Glu 85 90 95 Asn Ser Leu Ile Glu Phe Gln Leu AspGly Asn Asn Pro Tyr Asn Ile 100 105 110 Tyr Lys Ala Tyr Lys Ser Ser GlnAla Phe Asn Asn Glu Lys Asp Leu 115 120 125 Thr Asn Phe Thr Tyr Pro SerIle Asp Tyr Ile Ile Phe Leu Asp Ser 130 135 140 Asp Asn Tyr Trp Lys LeuAsn Cys Ile Glu Glu Cys Val Ile Arg Met 145 150 155 160 Lys Asn Val AspVal Leu Trp Phe Asp His Asp Cys Thr Tyr Glu Asp 165 170 175 Asn Ile LysAsn Lys His Lys Lys Thr Arg Met Glu Ile Phe Asp Phe 180 185 190 Lys LysGlu Cys Ile Ile Thr Pro Lys Glu Tyr Ala Asn Arg Ala Leu 195 200 205 SerVal Gly Ser Arg Asp Ile Ser Phe Gly Trp Asn Gly Met Ile Asp 210 215 220Phe Asn Phe Leu Lys Gln Ile Lys Leu Lys Phe Ile Asn Phe Ile Ile 225 230235 240 Asn Glu Asp Ile His Phe Gly Ile Ile Leu Phe Ala Ser Ala Asn Lys245 250 255 Ile Tyr Val Leu Ser Gln Lys Leu Tyr Leu Cys Arg Leu Arg AlaAsn 260 265 270 Ser Ile Ser Asn His Asp Lys Lys Ile Thr Lys Ala Asn ValSer Glu 275 280 285 Tyr Phe Lys Asp Ile Tyr Glu Thr Phe Gly Glu Asn AlaLys Glu Ala 290 295 300 Lys Asn Tyr Leu Lys Ala Ala Ser Arg Val Ile ThrAla Leu Lys Leu 305 310 315 320 Ile Glu Phe Phe Lys Asp Gln Lys Asn GluAsn Ala Leu Ala Ile Lys 325 330 335 Glu Thr Phe Leu Pro Cys Tyr Ala LysLys Ala Leu Met Ile Lys Lys 340 345 350 Phe Lys Lys Asp Pro Leu Asn LeuLys Glu Gln Leu Val Leu Ile Lys 355 360 365 Pro Phe Ile Gln Thr Lys LeuPro Tyr Asp Ile Trp Lys Phe Trp Gln 370 375 380 Lys Ile Lys Asn Ile 38535 346 PRT Campylobacter jejuni sialic acid synthase from C. jejuniOH4384 (ORF 8a of lipooligosaccharide (LOS) biosynthesis locus) 35 MetLys Glu Ile Lys Ile Gln Asn Ile Ile Ile Ser Glu Glu Lys Ala 1 5 10 15Pro Leu Val Val Pro Glu Ile Gly Ile Asn His Asn Gly Ser Leu Glu 20 25 30Leu Ala Lys Ile Met Val Asp Ala Ala Phe Ser Thr Gly Ala Lys Ile 35 40 45Ile Lys His Gln Thr His Ile Val Glu Asp Glu Met Ser Lys Ala Ala 50 55 60Lys Lys Val Ile Pro Gly Asn Ala Lys Ile Ser Ile Tyr Glu Ile Met 65 70 7580 Gln Lys Cys Ala Leu Asp Tyr Lys Asp Glu Leu Ala Leu Lys Glu Tyr 85 9095 Thr Glu Lys Leu Gly Leu Val Tyr Leu Ser Thr Pro Phe Ser Arg Ala 100105 110 Gly Ala Asn Arg Leu Glu Asp Met Gly Val Ser Ala Phe Lys Ile Gly115 120 125 Ser Gly Glu Cys Asn Asn Tyr Pro Leu Ile Lys His Ile Ala AlaPhe 130 135 140 Lys Lys Pro Met Ile Val Ser Thr Gly Met Asn Ser Ile GluSer Ile 145 150 155 160 Lys Pro Thr Val Lys Ile Leu Leu Asp Asn Glu IlePro Phe Val Leu 165 170 175 Met His Thr Thr Asn Leu Tyr Pro Thr Pro HisAsn Leu Val Arg Leu 180 185 190 Asn Ala Met Leu Glu Leu Lys Lys Glu PheSer Cys Met Val Gly Leu 195 200 205 Ser Asp His Thr Thr Asp Asn Leu AlaCys Leu Gly Ala Val Ala Leu 210 215 220 Gly Ala Cys Val Leu Glu Arg HisPhe Thr Asp Ser Met His Arg Ser 225 230 235 240 Gly Pro Asp Ile Val CysSer Met Asp Thr Gln Ala Leu Lys Glu Leu 245 250 255 Ile Ile Gln Ser GluGln Met Ala Ile Met Arg Gly Asn Asn Glu Ser 260 265 270 Lys Lys Ala AlaLys Gln Glu Gln Val Thr Ile Asp Phe Ala Phe Ala 275 280 285 Ser Val ValSer Ile Lys Asp Ile Lys Lys Gly Glu Val Leu Ser Met 290 295 300 Asp AsnIle Trp Val Lys Arg Pro Gly Leu Gly Gly Ile Ser Ala Ala 305 310 315 320Glu Phe Glu Asn Ile Leu Gly Lys Lys Ala Leu Arg Asp Ile Glu Asn 325 330335 Asp Thr Gln Leu Ser Tyr Glu Asp Phe Ala 340 345 36 352 PRTCampylobacter jejuni enzyme involved in sialic acid biosynthesis from C.jejuni OH4384 (ORF 9a of lipooligosaccharide (LOS) biosynthesis locus)36 Met Tyr Arg Val Gln Asn Ser Ser Glu Phe Glu Leu Tyr Ile Phe Ala 1 510 15 Thr Gly Met His Leu Ser Lys Asn Phe Gly Tyr Thr Val Lys Glu Leu 2025 30 Tyr Lys Asn Gly Phe Lys Asn Ile Tyr Glu Phe Ile Asn Tyr Asp Lys 3540 45 Tyr Phe Ser Thr Asp Lys Ala Leu Ala Thr Thr Ile Asp Gly Phe Ser 5055 60 Arg Tyr Val Asn Glu Leu Lys Pro Asp Leu Ile Val Val His Gly Asp 6570 75 80 Arg Ile Glu Pro Leu Ala Ala Ala Ile Val Gly Ala Leu Asn Asn Ile85 90 95 Leu Val Ala His Ile Glu Gly Gly Glu Ile Ser Gly Thr Ile Asp Asp100 105 110 Ser Leu Arg His Ala Ile Ser Lys Leu Ala His Ile His Leu ValAsn 115 120 125 Asp Glu Phe Ala Lys Arg Arg Leu Met Gln Leu Gly Glu AspGlu Lys 130 135 140 Ser Ile Phe Ile Ile Gly Ser Pro Asp Leu Glu Leu LeuAsn Asp Asn 145 150 155 160 Lys Ile Ser Leu Asn Glu Ala Lys Lys Tyr TyrAsp Ile Asn Tyr Glu 165 170 175 Asn Tyr Ala Leu Leu Met Phe His Pro ValThr Thr Glu Ile Thr Ser 180 185 190 Ile Lys Asn Gln Ala Asp Asn Leu ValLys Ala Leu Ile Gln Ser Asn 195 200 205 Lys Asn Tyr Ile Val Ile Tyr ProAsn Asn Asp Leu Gly Phe Glu Leu 210 215 220 Ile Leu Gln Ser Tyr Glu GluLeu Lys Asn Asn Pro Arg Phe Lys Leu 225 230 235 240 Phe Pro Ser Leu ArgPhe Glu Tyr Phe Ile Thr Leu Leu Lys Asn Ala 245 250 255 Asp Phe Ile IleGly Asn Ser Ser Cys Ile Leu Lys Glu Ala Leu Tyr 260 265 270 Leu Lys ThrAla Gly Ile Leu Val Gly Ser Arg Gln Asn Gly Arg Leu 275 280 285 Gly AsnGlu Asn Thr Leu Lys Val Asn Ala Asn Ser Asp Glu Ile Leu 290 295 300 LysAla Ile Asn Thr Ile His Lys Lys Gln Asp Leu Phe Ser Ala Lys 305 310 315320 Leu Glu Ile Leu Asp Ser Ser Lys Leu Phe Phe Glu Tyr Leu Gln Ser 325330 335 Gly Glu Phe Phe Lys Leu Asn Thr Gln Lys Val Phe Lys Asp Ile Lys340 345 350 37 221 PRT Campylobacter jejuni CMP-sialic acid synthetasefrom C. jejuni OH4384 (ORF 10a of lipooligosaccharide (LOS) biosynthesislocus) 37 Met Ser Leu Ala Ile Ile Pro Ala Arg Gly Gly Ser Lys Gly IleLys 1 5 10 15 Asn Lys Asn Leu Val Leu Leu Asn Asn Lys Pro Leu Ile TyrTyr Thr 20 25 30 Ile Lys Ala Ala Leu Asn Thr Lys Ser Ile Ser Lys Val ValVal Ser 35 40 45 Ser Asp Ser Asp Glu Ile Leu Asn Tyr Ala Lys Ser Gln AsnVal Asp 50 55 60 Ile Leu Lys Arg Pro Ile Ser Leu Ala Gln Asp Asn Thr ThrSer Asp 65 70 75 80 Lys Val Leu Leu His Ala Leu Lys Phe Tyr Lys Asp TyrGlu Asp Val 85 90 95 Val Phe Leu Gln Pro Thr Ser Pro Leu Arg Thr Asn IleHis Ile Asp 100 105 110 Glu Ala Phe Asn Leu Tyr Lys Asn Ser Asn Ala AsnAla Leu Ile Ser 115 120 125 Val Ser Glu Cys Asp Asn Lys Ile Leu Lys AlaPhe Val Cys Asn Glu 130 135 140 Tyr Gly Asp Leu Ala Gly Ile Cys Asn AspGlu Tyr Pro Phe Met Pro 145 150 155 160 Arg Gln Lys Leu Pro Lys Thr TyrMet Ser Asn Gly Ala Ile Tyr Ile 165 170 175 Leu Lys Ile Lys Glu Phe LeuAsn Asn Pro Ser Phe Leu Gln Ser Lys 180 185 190 Thr Lys His Phe Leu MetAsp Glu Ser Ser Ser Leu Asp Ile Asp Cys 195 200 205 Leu Glu Asp Leu LysLys Ala Glu Gln Ile Trp Lys Lys 210 215 220 38 277 PRT Campylobacterjejuni acetyltransferase from C. jejuni OH4384 (ORF 11a oflipooligosaccharide (LOS) biosynthesis locus) 38 Met Glu Lys Ile Thr LeuLys Cys Asn Lys Asn Ile Leu Asn Leu Leu 1 5 10 15 Lys Gln Tyr Asn IleTyr Thr Lys Thr Tyr Ile Glu Asn Pro Arg Arg 20 25 30 Phe Ser Arg Leu LysThr Lys Asp Phe Ile Thr Phe Pro Leu Glu Asn 35 40 45 Asn Gln Leu Glu SerVal Ala Gly Leu Gly Ile Glu Glu Tyr Cys Ala 50 55 60 Phe Lys Phe Ser AsnIle Leu His Glu Met Asp Ser Phe Ser Phe Ser 65 70 75 80 Gly Ser Phe LeuPro His Tyr Thr Lys Val Gly Arg Tyr Cys Ser Ile 85 90 95 Ser Asp Gly ValSer Met Phe Asn Phe Gln His Pro Met Asp Arg Ile 100 105 110 Ser Thr AlaSer Phe Thr Tyr Glu Thr Asn His Ser Phe Ile Asn Asp 115 120 125 Ala CysGln Asn His Ile Asn Lys Thr Phe Pro Ile Val Asn His Asn 130 135 140 ProSer Ser Ser Ile Thr His Leu Ile Ile Gln Asp Asp Val Trp Ile 145 150 155160 Gly Lys Asp Val Leu Leu Lys Gln Gly Ile Thr Leu Gly Thr Gly Cys 165170 175 Val Ile Gly Gln Arg Ala Val Val Thr Lys Asp Val Pro Pro Tyr Ala180 185 190 Ile Val Ala Gly Ile Pro Ala Lys Ile Ile Lys Tyr Arg Phe AspGlu 195 200 205 Lys Thr Ile Glu Arg Leu Leu Lys Ile Gln Trp Trp Lys TyrHis Phe 210 215 220 Ala Asp Phe Tyr Asp Ile Asp Leu Asn Leu Lys Ile AsnGln Tyr Leu 225 230 235 240 Asp Leu Leu Glu Glu Lys Ile Ile Lys Lys SerIle Ser Tyr Tyr Asn 245 250 255 Pro Asn Lys Leu Tyr Phe Arg Asp Ile LeuGlu Leu Lys Ser Lys Lys 260 265 270 Ile Phe Asn Leu Phe 275 39 270 PRTCampylobacter jejuni glycosyltransferase from C. jejuni OH4384 (ORF 12aof lipooligosaccharide (LOS) biosynthesis locus) 39 Met Pro Gln Leu SerIle Ile Ile Pro Leu Phe Asn Ser Cys Asp Phe 1 5 10 15 Ile Ser Arg AlaLeu Gln Ser Cys Ile Asn Gln Thr Leu Lys Asp Ile 20 25 30 Glu Ile Leu IleIle Asp Asp Lys Ser Lys Asp Asn Ser Leu Asn Met 35 40 45 Val Leu Glu PheAla Lys Lys Asp Pro Arg Ile Lys Ile Phe Gln Asn 50 55 60 Glu Glu Asn LeuGly Thr Phe Ala Ser Arg Asn Leu Gly Val Leu His 65 70 75 80 Ser Ser SerAsp Phe Ile Met Phe Leu Asp Ser Asp Asp Phe Leu Thr 85 90 95 Pro Asp AlaCys Glu Ile Ala Phe Lys Glu Met Lys Lys Gly Phe Asp 100 105 110 Leu LeuCys Phe Asp Ala Phe Val His Arg Val Lys Thr Lys Gln Phe 115 120 125 TyrArg Phe Lys Gln Asp Glu Val Phe Asn Gln Lys Glu Phe Leu Glu 130 135 140Phe Leu Ser Lys Gln Arg His Phe Cys Trp Ser Val Trp Ala Lys Cys 145 150155 160 Phe Lys Lys Asp Ile Ile Leu Lys Ser Phe Glu Lys Ile Lys Ile Asp165 170 175 Glu Arg Leu Asn Tyr Gly Glu Asp Val Leu Phe Cys Tyr Ile TyrPhe 180 185 190 Met Phe Cys Glu Lys Ile Ala Val Phe Lys Thr Cys Ile TyrHis Tyr 195 200 205 Glu Phe Asn Pro Asn Gly Arg Tyr Glu Asn Lys Asn LysGlu Ile Leu 210 215 220 Asn Gln Asn Tyr His Asp Lys Lys Lys Ser Asn GluIle Ile Lys Lys 225 230 235 240 Leu Ser Lys Glu Phe Ala His Asp Glu PheHis Gln Lys Leu Phe Glu 245 250 255 Val Leu Lys Arg Glu Glu Ala Gly ValLys Asn Arg Leu Lys 260 265 270 40 25 DNA Artificial SequenceDescription of Artificial SequenceCJ42 primer in heptosyltransferase-IIused to amplify LPS core biosynthesis locus 40 gccattaccg tatcgcctaaccagg 25 41 25 DNA Artificial Sequence Description of ArtificialSequenceCJ43 primer in heptosyltransferase-I used to amplify LPS corebiosynthesis locus 41 aaagaatacg aatttgctaa agagg 25 42 41 DNAArtificial Sequence Description of Artificial SequenceCJ-106 3′ primerused to amplify and clone ORF 5a 42 cctaggtcga cttaaaacaa tgttaagaatatttttttta g 41 43 37 DNA Artificial Sequence Description of ArtificialSequenceCJ-157 5′ primer used to amplify and clone ORF 5a 43 cttaggaggtcatatgctat ttcaatcata ctttgtg 37 44 37 DNA Artificial SequenceDescription of Artificial SequenceCJ-105 3′ primer used to amplify andclone ORF 6a 44 cctaggtcga cctctaaaaa aaatattctt aacattg 37 45 39 DNAArtificial Sequence Description of Artificial SequenceCJ-133 5′ primerused to amplify and clone ORF 6a 45 cttaggaggt catatgttta aaatttcaatcatcttacc 39 46 41 DNA Artificial Sequence Description of ArtificialSequenceCJ-131 5′ primer used to amplify and clone ORF 7a 46 cttaggaggtcatatgaaaa aagttattat tgctggaaat g 41 47 41 DNA Artificial SequenceDescription of Artificial SequenceCJ-132 3′ primer used to amplify andclone ORF 7a 47 cctaggtcga cttattttcc tttgaaataa tgctttatat c 41 48 322PRT Campylobacter jejuni Campylobacter alpha-2,3-sialyltransferase I(Cst-I) from C. jejuni OH4384 48 Met Thr Arg Thr Arg Met Glu Asn Glu LeuIle Val Ser Lys Asn Met 1 5 10 15 Gln Asn Ile Ile Ile Ala Gly Asn GlyPro Ser Leu Lys Asn Ile Asn 20 25 30 Tyr Lys Arg Leu Pro Arg Glu Tyr AspVal Phe Arg Cys Asn Gln Phe 35 40 45 Tyr Phe Glu Asp Lys Tyr Tyr Leu GlyLys Lys Ile Lys Ala Val Phe 50 55 60 Phe Asn Pro Gly Val Phe Leu Gln GlnTyr His Thr Ala Lys Gln Leu 65 70 75 80 Ile Leu Lys Asn Glu Tyr Glu IleLys Asn Ile Phe Cys Ser Thr Phe 85 90 95 Asn Leu Pro Phe Ile Glu Ser AsnAsp Phe Leu His Gln Phe Tyr Asn 100 105 110 Phe Phe Pro Asp Ala Lys LeuGly Tyr Glu Val Ile Glu Asn Leu Lys 115 120 125 Glu Phe Tyr Ala Tyr IleLys Tyr Asn Glu Ile Tyr Phe Asn Lys Arg 130 135 140 Ile Thr Ser Gly ValTyr Met Cys Ala Ile Ala Ile Ala Leu Gly Tyr 145 150 155 160 Lys Thr IleTyr Leu Cys Gly Ile Asp Phe Tyr Glu Gly Asp Val Ile 165 170 175 Tyr ProPhe Glu Ala Met Ser Thr Asn Ile Lys Thr Ile Phe Pro Gly 180 185 190 IleLys Asp Phe Lys Pro Ser Asn Cys His Ser Lys Glu Tyr Asp Ile 195 200 205Glu Ala Leu Lys Leu Leu Lys Ser Ile Tyr Lys Val Asn Ile Tyr Ala 210 215220 Leu Cys Asp Asp Ser Ile Leu Ala Asn His Phe Pro Leu Ser Ile Asn 225230 235 240 Ile Asn Asn Asn Phe Thr Leu Glu Asn Lys His Asn Asn Ser IleAsn 245 250 255 Asp Ile Leu Leu Thr Asp Asn Thr Pro Gly Val Ser Phe TyrLys Asn 260 265 270 Gln Leu Lys Ala Asp Asn Lys Ile Met Leu Asn Phe TyrAsn Ile Leu 275 280 285 His Ser Lys Asp Asn Leu Ile Lys Phe Leu Asn LysGlu Ile Ala Val 290 295 300 Leu Lys Lys Gln Thr Thr Gln Arg Ala Lys AlaArg Ile Gln Asn His 305 310 315 320 Leu Ser 49 231 PRT Haemophilusinfluenzae putative ORF from GenBank #U32720 49 Met Gln Leu Ile Lys AsnAsn Glu Tyr Glu Tyr Ala Asp Ile Ile Leu 1 5 10 15 Ser Ser Phe Val AsnLeu Gly Asp Ser Glu Leu Lys Lys Ile Lys Asn 20 25 30 Val Gln Lys Leu LeuThr Gln Val Asp Ile Gly His Tyr Tyr Leu Asn 35 40 45 Lys Leu Pro Ala PheAsp Ala Tyr Leu Gln Tyr Asn Glu Leu Tyr Glu 50 55 60 Asn Lys Arg Ile ThrSer Gly Val Tyr Met Cys Ala Val Ala Thr Val 65 70 75 80 Met Gly Tyr LysAsp Leu Tyr Leu Thr Gly Ile Asp Phe Tyr Gln Glu 85 90 95 Lys Gly Asn ProTyr Ala Phe His His Gln Lys Glu Asn Ile Ile Lys 100 105 110 Leu Leu ProSer Phe Ser Gln Asn Lys Ser Gln Ser Asp Ile His Ser 115 120 125 Met GluTyr Asp Leu Asn Ala Leu Tyr Phe Leu Gln Lys His Tyr Gly 130 135 140 ValAsn Ile Tyr Cys Ile Ser Pro Glu Ser Pro Leu Cys Asn Tyr Phe 145 150 155160 Pro Leu Ser Pro Leu Asn Asn Pro Ile Thr Phe Ile Leu Glu Glu Lys 165170 175 Lys Asn Tyr Thr Gln Asp Ile Leu Ile Pro Pro Lys Phe Val Tyr Lys180 185 190 Lys Ile Gly Ile Tyr Ser Lys Pro Arg Ile Tyr Gln Asn Leu IlePhe 195 200 205 Arg Leu Ile Trp Asp Ile Leu Arg Leu Pro Asn Asp Ile LysHis Ala 210 215 220 Leu Lys Ser Arg Lys Trp Asp 225 230

1-42. (Cancelled)
 43. An isolated or recombinantly produced polypeptidecomprising a polypeptide having β1,3-galactosyltransferase activity,wherein the β1,3-galactosyltransferase polypeptide comprises an aminoacid sequence that is at least 80% identical to SEQ ID NO:29.
 44. Theisolated or recombinantly produced polypeptide of claim 43, wherein theamino acid sequence is at least about 90% identical to SEQ ID NO:29. 45.The isolated or recombinantly produced polypeptide of claim 43, whereinthe amino acid sequence is at least about 95% identical to SEQ ID NO:29.46. The isolated or recombinantly produced polypeptide of claim 43,wherein the an amino acid sequence is SEQ ID NO:29.
 47. The isolated orrecombinantly produced polypeptide of claim 43, further comprising a tagfor purification.
 48. The isolated or recombinantly produced polypeptideof claim 43, wherein the polypeptide is recombinantly produced and atleast partially purified.
 49. The isolated or recombinantly producedpolypeptide of claim 43, wherein the polypeptide is expressed by aheterologous host cell.
 50. The isolated or recombinantly producedpolypeptide of claim 49, wherein the host cell is E. coli.